- Email: [email protected]
Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium
Biol Cell (1994) 81,95-I 19
95
© Elsevier, Pads
Original article
Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium Anne-Marie Callen a, Andr6 Adoutte a, Jose Manuel Andrew b, A n n e Baroin-Tourancheau a, Marie-H61~ne Br6 a, Purificacion Calvo Ruiz e, Jean-Claude C16rot a, Pilar Delgado a, A n n e Fleury a, Rachel Jeanmaire-Wolf a, Vladimir Viklicky d, Eduardo Villalobo c, Nicolette Levilliers a aLaboratoire de Biologie Cellulaire 4, URA l134-CNRS, Bdtiment 444, Universitd de Paris-Sud, 91405 Orsay Cedex, France; b Centro de Investigaciones Biologicas, c/o Velazques 144, 28006 Madrid; bDepartarnento de Microbiologia, Facultad de Biologia, Universidad de Sevilla, Apdo 1095, 41080 Sevilla, Spain; dlnstitute of Molecular Genetics, Czechoslovak Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic (Received 28 June 1994; accepted 18 July 1994)
Summary - Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and I0 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the t~ N-terminal domain, one in the fl C-terminal one, and two in tx and fl central stretches). The remaining ones decorated only a specific subset of rnicrotubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of wtubulin whereas the epitopes of three other ones (TAP 952 °, AXO 58 and AXO 49 °) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both ix- and fl-tubulins. The AXO 49 ° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences. ciliate tubulin / axonemal tubulin / tubulin epitopes / tubulin post-translational modifications / immunofluorescence
Introduction Two recurrent questions in microtubule research have been those of the origin of tx- and.fl-tubulin diversity and its functional significance. Concerning the first question, much progress has been made during the last few years with the realization that tubulin diversity can be attributed both to primary genetic redundancy of tubulin coding genes and to a variety of post-translational modifications. The inventory of both gene diversity and the biochemical bases of post-translational modifications has advanced in a variety of organisms, especially metazoans and metaphytes, but also in several protists and fungi (see [12, 33, 35, 41, 55, 64, 66] for reviews). For metazoans, the picture usually emerging has been that of a fairly high diversity of genes coding for a variety of isotypes often expressed in a tissue-specific pattern coupled to at least five well identified post-translational modifications: acetylation of wtubulin on lysine 40 [23], phosphorylation of a fl-tubulin isotype near the C-terminal end [3, 22, 25, 28, 31, 48], polyglutamylation of both ct- and fl-tubulins near the C-terminal extremity [3, 24, 57, 60], detyrosination of the C-terminal end of t~-tubulin [7, 56, 69]
and removal of the glutamyl tyrosine dipeptide at the C-terminal end of tx-tubulin [52]. In protists, especially in ciliated or flagellated species, one m a j o r difference has been observed in terms of genetic redundancy with respect to multiceUular organisms: a- and fl-tubulins are each encoded by a very small number of extremely similar (if not identical) genes (at the amino-acid level). A striking example is that of the ciliate Tetrahymena which builds its large variety of microtubular networks from only one a and two almost (Tpyriformis [6]) or completely (T thermophila [29]) identical fl gene products. In contrast, post-translational modifications in protists appear to be very similar to those identified in multicellular organisms: in particular acetylation has been discovered in one protist [44, 46, 47] and then extended to a variety of protists and other organisms (reviewed in [45]). As for glutamylation, it has just been discovered in ciliates [10]. In addition, one notable, still biochemically uncharacterized post-translational modification has been found to occur in Paramecium axonemes and later in those of a very large array of organisms extending from protists to mammals [1, 2]. Except for trypanosomes [61], evidence for detyrosination is lacking at the moment in other protists.
96
AM Callcn et al
Concerning the second question, unequivocal demonstrations of the function of microtubule primary and secondary diversity are scarce to date ([37], for a review see [40]). Ciliates and specially Paramecium are well adapted for analysis of the processes through which diversification of initially identical microtubules takes place within the same cytoplasm. Indeed, at least 13 distinct microtubular arrays have been described in Paramecium [15], and although the number of tx- and fl-tubulin genes is higher in Paramecium than in Tetrahymena (respectively four and three), all the sequence work carded out so far shows that the resulting proteins are e x t r e m e l y similar [20, 21]. Thus in this system, most if not all, differences in the properties of the diverse microtubular arrays can be attributed to post-translational modifications and/or interactions with accessory proteins. So far, the immunocytochemical analysis of microtubule diversity in Paramecium [2, 10, 15, 70] has mostly relied on well characterized antibodies raised in other systems such as the 'universal' anti c~- and anti-fl-tubulin antibodies D M 1 A and DM1B [9] reacting respectively on tx- and fl-tubulin primary sequences [11], or antibodies directed against well identified post-translational modifications such as 6-11B-1 which reacts with acetylated tx-tubulin [53], or GT 335 which recognizes glutamylated tubulin [75]. In addition, a polyclonal serum raised against Paramecium axonemal tubulin [14] was used and it was recently suggested that its major epitope corresponds to a post-translational modification different from others [2]. In the present work, we have approached the question of microtubule diversity in Paramecium by directly isolating libraries of monoclonals to ciliate tubulins, aiming at recovering as large a spectrum as possible. We describe the various fusions carried out and the characterization of eight monoclonal antibodies and 10 hybridoma supernatants in terms of specificity over a- and fl-tubulins, interaction with non-ciliate tubulins, location of the epitope as deduced from reactivity on peptide maps and from sequence comparisons between ciliate and vertebrate tx- and fl- tubulins, and, finally, identification of the epitope as being located in the primary sequence of tubulin or being due to a post-translational modification. This last distinction was made through the presence or absence of recognition over fusion proteins generated by genetic engineering in E coli: it was based on the idea that most (if not all) of the modifications known to occur, on tubulin do not take place in E coli and even if they did, it would be highly unlikely that the E coli modification enzymes would recognize a typically eukaryotic protein such as tubulin. We also document the irnmunocytochemicai properties of the various antibodies. It will be seen that a very broad collection of antibodies has been obtained, apparently revealing an extensive microtubular diversity existing in Paramecium; these antibodies will provide powerful tools for analysis of both the nature and genesis of post-translational modifications and of the patterning of microtubules during morphogenesis.
Materials and methods Strains and cell cultures Paramecium tetraurelia cells (strain d4-2) were grown at 27°C in phosphate buffered Wheat Grass Powder infusion, supplemented with 0.4/2g/ml fl-sitosterol and baeterized with Klebsiella pneumoniae. Euplotes aediculatus and Stylonychia lemnae (a generous gift from Drs E Helftenbein and M Schlegel)
were fed with Tetrahymena cells grown axenically according to Fleury [26]. 3T3 cells were cultured in RPMI medium supplemented with 2 mM glutamine, 100 IU/ml penicillin and 100 /ag/ml streptomycin, then grown on glass coverslips for immunofluorescence. PtK2 epithelial cells were grown as described by Geuens et al [32]. Antigens Cytoskeletal tubulin of Euplotes Euplotes cells were treated in PHEM buffer, at pH 6.9 [62], supplemented with 0.5 Triton X-100, leupeptin (4/zM') and PMSF (0.3 mM) for 5 rain. Extracted cells were boiled for 3 min in electrophoresis sample buffer and loaded onto 7.5-15% acrylamide gradient, 12-cm long, 3-mm thick, preparative SDS-polyacrylamide gels [43]. After electrophoresis, tubulin was visualized after a 0.25 M KCI treatment of the gels and the acrylamide bands were excised; tubulin was electroeluted in a 0.025 M TrisHCI, 0.192 M glycine buffer at pH 8.3 containing 0.025% SDS, using a Biotrap apparatus from Schleicher and Schuell (Ecquevilly, France). It was kept frozen at -200C. Paramecium axonemes Prepared according to Geuens et al [32] and kept frozen at -80°C. Axonemal tubulin of Paramecium Axonemes were submitted to preparative SDS-PAGE. Tubulin was electroeluted and stored as described for Euplotes. Production of monoclonal antibodies
BALB/c mice immunizations, fusions, hybridoma screening and sub-cloning were carded out as described by Jeanmaire-Wolf et al [39] with minor modifications as detailed in table I. ELISA development was made using either a secondary sheep antimouse IgG antibody coupled to peroxidase (Diagnostics Pasteur, France) and, as substrate, 0.1% ortho-phenylene diamine with 0.002% 1-I202or a rabbit anti-mouse IgG labeled with fl-galaetosidase (Biosys, Compi~gne, France) and ortho-nitrophenyl-fl-ogalactoside (Sigma, St Louis, MO, USA) as chromogen. Protein preparations Construction offusion proteins Construction of fl-galactosidase a-tubulin fusion proteins. A recombinant plasmid (pBR 322) containing the c(2-tubulin gent from Stylonychia lemnae [36] was kindly provided by F Caron (Ecole Normale Sup6rieure, Paris, France). Different portions of the a-tubulin gene were cloned in-frame behind DNA encoding fl-galactosidase in the pEX expression vectors (Boehringer, Mannheim, Germany). The coding sequence of this gene contains the codon UAA (position 177) which codes for glutamine in many ciliates [65]. Expression of the inserts containing this codon gives thus truncated fusion proteins. The different hybrid fl-galactosidase constructions were designed so that the STOP codon was never located upstream of the insert to be expressed. The expressed inserts correspond to the following domains of the a-tubulin protein: one segment located towards the N-terminal side of Stylonychia ~-tubulin, N, (amino-acids 22-176) and another one comprising the C-terminal side, C (21A. 445) (fig 3). To express the internal domain (177-214), a distinct construction experiment has been performed. Expression of the hybrid proteins. Expression of the hybrid fl-galactosidase is under the control of a ,t.-Pr promoter. Transformations were performed using pop 2136, a host strain carrying the temperature sensitive lambda CI repressor allele. Recombinant plasmids were amplified at 30°C and their transient expression was induced by shifting to 42"C. After 1 or 2 h at 42*C, 1 ml of the culture was removed. The pelleted bacteriai cells were resuspended in sample buffer, boiled for 5 min and submitted to SDS-PAGE for Western blot analysis.
Tubulin diversity in Paramecium
97
Table I. Overview of immunizations and fusion results. The names which are indicated are those of antibodies which have been chosen
for this study; their characteristics are recapitulated in table II.
Name offusion Antigen
Interval between boosts
TEU
Electroeluted Euplotes Electroeluted Paramecium cytoskeletal tubulin axonemal tubulin (tubulin from (tubulin from Euplotes = TEU) axonemes of Paramecium = TAP) 3 weeks 10 weeks 3 weeks 20 weeks + 4 days: 1 fusion + 3 days: 1 fusion
Number of hybridoma containing wells tested Number (and percent) of positive ELISA tests
Tests carried out and criteria for storing Major characteristics of hybridoma supernatant tested; decoration of: - all microtubular networks - stable microtubules - cilia along all their length and all microtubular networks - cilia, except at their tip
AXO Paramecium axonemes (= AXO) 1 week 4 weeks + 4 days: 1 fusion
864
480
480
118 (13.6%)
65 (13.5%)
91 (18.9%)
1. ELISA on Euplotes, - ELISA on Paramecium - ELISA on axonemes, Euplotes Paramecium, pig brain tubulin and pig brain tubulin and pig brain tubulin hybridoma retained if strongly or moderately posititve in ELISA on Euplotes and/or Paramecium tubulin 2. Immunofluorescence over whole cells of Paramecium; ~ hybridomas stored if: - clear decoration a - decoration of cilia of various microtubular networks b - various decoration patterns of intracellular networks Number Name Number Name Number Name 19 2 0
TEU 435*, TEU 310 °, TEU 441, TEU 4211G, TEU 4211H, TEU 4312 TEU 318 °, TEU 348 °
0
Expression of a fl-galactosidase-ot-tubulin fusion protein containing the domain (177-214). A copy of the ¢x-tubulin gene from Euplotes aediculatus was amplified using a couple of degenerate primers. The amplification product, 1.2 kb long, was cloned blunt ended in the phagemid pBS (Stratagene, La Jolla, CA, USA) in-frame with the DNA encoding the N-terminal part of the ~-galactosidase. In Euplotes aediculatus the sequence of the gene does not contain any STOP codon since this species appears to use the u n i v e r s a l code [65]. In this case, the expressed insert corresponds to a large fragment covering the almost complete sequence of the protein, E (25--412) (fig 3). Expression by this plasmid is controlled through a promoter that is activated with isopropyl-/~-D-thiogalaetopyranoside (IPTG). In the strain XL1 blue transformed with the recombinant plasmid, induction was induced by addition of 5 mM IPTG. After a 4-h incubation in presence of IPTG, the bacterial cells were treated as described above.
Cell extracts Stylonychia cytoskeletons.
TAP
4 1 2
TAP 9311, TAP 931B, TAP 926, TAP 956 ° TAP 931E TAP 952 °
0
4
AXO 16, AXO 45 °
5 0
AXO 58
3
A X O 49*
diluted to 1 mg/ml in 1 M sodium phosphate at pH 8 supplemented with 1 mM GTP and 1 mM MgCI2; acetic anhydride was added under constant stirring, at room temperature, by 2-/.d aliquots, every 5 rain while maintaining a constant pH (8-8.5) by addition of 1 N NaOH. The final acetic anhydride concentration was 1% and the whole procedure took 30 rain. Therefore, under these conditions, most e-aminogroups of lysine and also tyrosine residues should be acetylated. The latter can be deaeetylated by hydroxylamine at neutral pH. To this purpose, after dialysis of the sample, hydroxylamine in 20 mM sodium phosphate at pH 7 was brought to 0.1 M in the acetylated tubulin sample; this solution was kept under constant stirring during 30 rain and then dialyzed. Acetylated tubulin was frozen in liquid nitrogen.
Reference anti-tubulin antibodies Monoclonals
Stylonychia cells were treated with
DMIA and DM1B [9] were purchased from Amersham (Les Ulis, France); TU-01 [73] was used; 6-11B-1 culture supematant [53] was a gift of Dr G Piperno (The Rockefeller University, New York, USA) whereas 6-11B-I ascitic fluid was purchased from Sigma; 111 B52 C2 [74] and GT 335 [75] were generously provided by Dr A Wolff (Coll~ge de France, Paris).
0.5% Triton X-100 in PHEM buffer supplemented with 4/zM leupeptin for 5 rain. Extracted cells were boiled in electrophoresis sample buffer and kept frozen in aliquots at -80°C. PtK2 cells. Confluent PtK2 cells were rinsed with PBS, then scrapped from Petri dishes, and boiled for 5 min in electrophoresis sample buffer supplemented with 8/AM leupeptin and 2 mM PMSF and kept frozen at -20°C. Paramecium cytoplasmic extract. Prepared according to Br6 et al [I0]. Phosphocellulose purified porcine brain tubulin. Kindly provided by Dr MF Carlier (CNRS, Gif-sur-Yvette, France).
Immunofluorescence microscopy
Chemical acetylation of porcine brain tubulin. Acetylation was carried out according to Riordan and Vallee [58, 59] and Piperno and Fuller [53] with some modifications: tubulin was
For Paramecium, the immunofluorescence protocol described in Jeanmaire-Wolf et al [39] was followed with minor modifications: the permeabilization buffer (PHEM at pH 6.9 with 1% Tri-
Polyclonals Affinity-purified C 98, C 140 and C 113 monospecific IgG antibodies [5] were employed.
98
A_M Callen et al
ton X-100) was supplemented with leupeptin (4/tM) and the ghosts were fixed in freshly dissolved 2% paraformaldehyde, in PHEM buffer for 45 min. After washes, they were incubated in one anti-tubulin antibody and then in the fluorescein-conjugated sheep-anti-mouse IgG antibody (Diagnostics Pasteur, France) diluted 1:200 and rinsed. Cells were mounted using Citifluor (London) and examined on a Bio-Rad Lasersharp MRC 600 confocal microscope equipped with an argon laser. For 3T3 and PtK2 cells, two fixation procedures were used: either with methanol at -20°C for 3 rain or with 1/10 formaldehyde (commercial solution) for 30 min in PBS followed by 50 mM NH4CI in PBS (2 × 5 rain) and 0.1% Triton X-100 in PBS (10 rain). Cells were rinsed in PBS, saturated with 3% BSAPBS buffer, incubated with primary antibodies diluted in 3% BSA-PBS for 90 rain, further incubated with fluorescein-conjugated sheep-anti-mouse IgG (1:50 in 3% BSA-PBS for 30 rain) and processed like Paramecium cells. Gel electrophoresis and immunoblotting SDS-PAGE was conducted according to Laemmli [43]. Briefly, proteins were boiled for 3 rain in Laemmli's sample buffer and loaded on 0.75-ram thick, 10% or 8% polyacrylamide mini-gels (unless otherwise mentioned) containing a lower gel buffer respectively adjusted to pH 8.2 or pH 9.3 for Paramecium axonemes and fusion proteins or porcine brain tubulin. SDS from BDH Laboratory Supplies (Poole, UK) was mostly used [1]. After electrophoresis, gels were either stained by Coomassie brilliant blue R 250 or electro-transferred onto 0.45//m nitrocellulose filters using a semi-dry transfer CarboGlas apparatus (Schleicher and Schuell) and the protocol described by Kyhse-Andersen [42] (with methanol replaced by isopropanol in the transfer buffers). Membranes were stained With Ponceau red, destained, saturated in PBS-3% BSA for 1 h at 37°C, further incubated for 1 h at 37°C (or overnight at room temperature) with primary antibodies diluted in PBS-0.25% BSA-0.1% Tween 20, and extensively washed in the antibody buffer. For IgG detection, filters were incubated for 1 h at room temperature with an alkaline phosphatase-labeled goat anti-mouse IgG at a dilution of 1:7500 (Promega, Madison, WI, USA) or with a peroxidase-labeled sheep anti-mouse IgG secondary antibody at 1:2000 (Diagnostics Pasteur). For IgM detection, two successive incubations in class-specific secondary antibody then in labeled enzyme, were used. They involved biotlnylated goat anti-mouse IgM at 1:200 (Amersham), followed by alkaline phosphatase-conjugated streptavidin at 1:1000 (Immunotech, Marseille, France). Development of alkaline phosphatase was carried out with NBT (nitro blue tetrazolium) and BCIP (5-bromo-4-chloro-3indolyl phosphate) as substrates (provided by Sigma). Development of peroxidase was performed with the ECL system from Amersham. Dot-blotting Antigens (2/11 in Laemmli's sample buffer) were dotted on 0.45 p.m nitrocellulose, the membrane was air-dried and saturated in PBS-3% BSA, then treated with antibodies similarly as after electrophoresis and electro-transfer. Peptide mapping In a first step, ~x- and/3-tubulin bands from 10 to 20/~g Paramecium axonemes were separated then cut out from Coomassie blue-stained 12-cm long, 0.75-ram thick 8% polyacrylamide gels [43] performed: i) either, at pH 8.25, with 0.1% specially pure (98-99%) SDS from BDH; or ii) at pH 9.25, with 0.1% SDS from Sigma (70% pure). The respective running buffers contained the corresponding SDS. In the presence of pure SDS at pH 8.25, Paramecium a-tubulin migrates faster than ~tubulin as reported for Tetrahymena tubulin [67], whereas, at pH 9.25, both subunits comigrate. However, they are resolved with impure SDS at pH 9.25 but their relative migrations are reversed and become similar to those of brain tubhlin subunits. This altered behaviour is probably due to the contaminants of SDS, myristil (C14) and eetyl (C16) sulfates, which have been shown to achieve a good separation of brain a- and fl-tubulins [8].
In a second step, the tubulin bands (kept at - 2 0 ° C ) were loaded in the wells of an 1l-cm long, l-ram thick resolving gel made of a 15-20% acrylamide gradient in the presence of BDH SDS: i) either at pH 8.25, in standard Laemmli conditions (figs 6A, 9, 10 and 12); or ii) at pH 8.8, by doubling the "Iris concentration in the lower gel and running buffers, as described by Fling and Gregerson [27] to improve the resolution of small peptides (fig 6B). The tubulin bands were submitted to mild proteolysis by Staphylococcus aureus V8 protease, type XVII-B (Sigma), in the presence of 0.3% 2-mereaptoethanol, during a 2-h migration in the stacking gel (3 cm long) followed by a 1-h arrest of proteins at the interface of the stacking and resolving gels (slightly modified after Cleveland et al [13]). Rainbow coloured protein molecular mass markers (range 2.35-46 kDa) were from Amersham. After overnight electrophoresis, the gels were silver stained according to Merril et al [50], followed by intensification adapted from Merril et al [51]. Briefly, the silver coloured gels were completely destained by soaking for 5-20 rain in a 0.5% solution of Lumiere reducer (a photochemical reagent containing potassium ferricyanide), and after extensive washes in water, were restained by direct incubation in silver nitrate followed by development with AnalaR formaldehyde (BDH). Milli Q filtered water (Millipore, Saint-Quentin-en-Yvelines, France) was used throughout. Alternatively, the gels were electro-transferred onto 0.1 /tin pore size nitrocellulose (Schleicher and Schuell) according to Towbin et al [71], except for methanol replaced by ethanol in the transfer buffer. After Ponceau red staining, destaining and saturation, the filters were incubated overnight in antibodies (diluted in the same solution as for immunoblotting), followed by a 1-h incubation in biotinylated goat anti-mouse IgG or biotinylated donkey anti-rabbit IgG at a dilution of 1:1600. After a further 1-h incubation in streptavidin-biotinylated horseradish peroxidase complex at 1:1 600, detection was achieved using the ECL system. All incubation steps were performed at room temperature and separated by exhaustive 1-h rinsings as for immunoblotting. Antibodies were provided by Amersham.
R ~ Overview o f immunizations and fusions results (tables I, II) The objectives of the successive fusions differed (table I). Initially, we were interested in recovering the full diversity of possible antibodies to tubulin and thus used cytoskeletal tubulin o f E u p l o t e s as i m m u n o g e n in which a m i x t u r e o f T r i t o n - r e s i s t a n t c y t o p l a s m i c , c o r t i c a l and axonemal tubulins is expected to be present; this corresponds to fusion TEU. In the two later fusions, we were specifically interested in recovering monoclonals yielding similar decorations o f cilia and stable microtubules as those given by a rabbit p o l y c l o n a l serum obtained by injecting P a r a m e c i u m a x o n e m a l tubulin in the form of acrylamid bands [2, 14]. Therefore, either electroeluted P a r a m e c i u m a x o n e m a l tubulin (fusion T A P ) or w h o l e a x o n e m e s w i t h o u t any e l e c t r o p h o r e t i c purification o f tubulin and denaturing step were also injected (fusion
~o). Ciliate tubulin, whatever the form under which it was injected to mice, proved to be immunogenic. Effectively, the sera of all mice tested (22) recognized tubulin after the third injection both on immunoblots and in immunofluorescence tests where it decorated a variety of microtubular networks and/or cilia o f P a r a m e c i u m and Euplotes. The percentage o f E L I S A positive h y b r i d o m a supernatants was respectively 13.6, 13.5 and 18.9%, a fairly high proportion considering that tubulin is an evolutionary conserved protein.
21
22
A
19 20
TAP 952 °
AXO58
A X O 49* GT 335*
6-11B-l* TEU 318 ° TEU 348 ° TAP 931E
T A P 956*
14
15 16 17 18
TAP 926G
DMIB* A X O 45 °
DMIA* TU01* TAP 9311 TAP 931B T E U 435* TEU 310 ° T E U 441 TEU 4211G TEU 4211H TEU 4312 A X O 16
name
13
2 12
3 4 5 6 7 8 9 10 11
1
and
IgG1
IgGl
IgG1 IgG1
IgG1 IgG1 IgG1 IgG1
IgG1
IgGl
IgG1 IgG1
IgG1 IgG 1 IgG1 IgG3 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgM
Type
ix+13
13
ix+13 ix+13
. . fl a n d / o r ixA
e e e e
ix + 13
ix + 13
ix + 13 13 a n d / o r ixA
ix ix ix ix
ix + ]3
ix + 13
fl 13
a a ix a ix ix a a a ix ix
Paramecium tubulin axonemal cytoplasmic
. .
.
-
-
-
+ + + + + + + + + +
.
.
.
-
e
e
-
+
.
.
.
-
-
-
+ + + + + + + + + +
lmmunobloting Fusion proteins E C N
.
.
•
ix
ix ix ix ix
ix+13
ix
fl 13
a ix ix ix ix ix
0
0
0 I
1/10 1/5 1/5 < 1/10
1/i0
1/10
1/i0
1 1 < 1/20 0 0 0 0 < 1/20 1/10
Pig brain tubulin Blot Dot reactivity
+ + + +
-
.
.
Ac Ac Ac Ac
+ +
+ . .
+
.
.
.
.
.
.
-
+
.
.
+ Ac
+ Ac
-
+ +
-
-
+ + -
+
.
.
.
.
+
+
+
-
+
-
-
-
. + +
-
+
+ +
+ + -
+ + + +
+
ix ( 1 5 9 - 2 8 1 ) fl ( 1 8 8 - 3 1 0 ) ix ( 1 5 9 - 2 8 1 ) 13(188-310) ix ( L y s 40)* ix ( 1 - 1 0 0 ) ix ( 1 - 1 0 0 ) ix ( 1 - 1 0 0 )
13 ( 4 1 6 - 4 3 0 ) * 13 (257---442)
ix (426--430)* tx ( 6 5 - 9 7 ) * ix ( 9 8 - 1 3 6 ) ix ( 9 8 - 1 3 6 ) a (98-136) ix ( 9 8 - 1 3 6 ) a (98-136) ix ( 9 8 - 1 3 6 ) ix ( 9 8 - 1 3 6 ) ix ( 9 8 - 1 3 6 )
Epitope location
ix (254 449) 13 (257--442)
13(257 4~2)
ix (Glu 445)* 13 (Glu 435 or 438)* ix (254 449)
Immunofluorescence lnterphasic metazoan cells 3T3 PtK2 Form-T Methanol Form-T Methanol
i n d i c a t e d in bold a n d d e s i g n a t e d b y °. * R e f e r e n c e antibodies, the e p i t o p e s o f w h i c h are well k n o w n o n v e r t e b r a t e t u b u l i n s ; literature r e f e r e n c e s : D M 1 A * , D M 1 B * [11]; T U - 0 1 * [44]; G T 3 3 5 * [3, 24, 57, 60, 75]. B l o t t i n g o n Paramecium t u b u l i n a n d f u s i o n proteins: e, v e r y w e a k s t a i n i n g (see [10]). F u s i o n p r o t e i n s : E, C, N; s e e s c h e m a t i c d i a g r a m (fig 3). l e g e n d to f i g u r e 2. I n t e r p h a s i c m e t a z o a n cells: F o r m - T , f o r m o l - T r i t o n o r m e t h a n o l i n d i c a t e t h e f i x a t i o n p r o c e d u r e ; +, s t r o n g l a b e l i n g ; - , w e a k , a s p e c i f i e l a b e l i n g or n o decoration; t u b u l i n a n t i b o d y typical labeling. E p i t o p e location: inferred f r o m reactivity o n p e p t i d e m a p s a n d f u s i o n p r o t e i n s (see fig 3).
B
Anti P T M C
Antiacetylated ix-tubulin: A c
Anti ix+13 tubulin sequence
Anti fltubulin s e q u e n c e : flS
Anti atubulin s e q u e n c e : ixS
Classification
n e d a n t i b o d i e s are [18, 34]; 6 - 1 1 B - 1 " D o t reactivity; s e e Ae, anti-aeetylated
Table I I . S u m m a r y o f t h e c h a r a c t e r i s t i c s o f t h e a n t i b o d i e s . D e n o m i n a t i o n s : ixS, flS, A c , P T M ( p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n ) . N u m b e r s 1, 2, etc, c o r r e s p o n d to t h o s e e m p l o y e d in f i g u r e 2. Sub-clo-
100
AM Callen et al •
x
i
•
p
°
.
.
°"
"x
-'..
:" "" ,."
• S" .. -D :., ,-I,
.~
:" ': '~,•" -
:.
',
'4'.:]:
-
2
,
% !I
.
.
.
.
Of the 18 antibodies described in this paper, eight were successfully sub-cloned; the others remained at the stage of hybridoma supernatant because antibody production was lost upon thawing or sub-cloning. They are mentioned nonetheless because they not only yield interesting results, but they also appear to be truly monoclonal by several criteria. All the antibodies belong to the IgG class except A X O 16, an IgM. All the IgGs belong to the IgG1 subclass except TAP'931B which belongs to the IgG3 one (table ID. In addition to immunocytochemical analysis on Paramecium and metazoan cells, the hybridoma supernatants were submitted to detailed characterization through immunoblotting and immunocytochemical experiments which allowed to group the antibodies into distinct categories presented below. The characteristics o f the eight monoclonal antibodies "(designated by an ° throughout the text) and the 10 hybridoma supematants are recapitulated in table II.
Fig 1. Two projections of a Paramecium decorated with the TEU 435 ° antibody (1:5) and observed by confocal microscopy, a. Ventral side. b. Dorsal side. The antibody decorates all the microtubules, ie the postoral fiber (Po) associated with the oral area (O), the microtubules associated with the contractive vacuole systems (large arrows), as well as the intracytoplasmic network which appears densely ramified (arrows). In addition, the tips of the cilia are strongly stained (arrowheads), while the rest of the axonemal structure is only faintly decorated. Scale bars, 25/.tm.
Antibodies reacting conspicuously with fusion proteins Anti-~-tubulin sequence (ctS) antibodies Nine of the monoclonal antibodies and hybridoma supematants proved to be positive on fusion proteins indicating that they are directed against an tx-tubulin sequence determinant; they were characterized in detail (table II): T A P 9311, TAP 931B, T E U 435 °, TEU 310 °, T E U 441, T E U 4211G, TEU 4211H, T E U 4312, and A X O 16. In immunofluorescence tests carried out on whole ceils o f P a r a m e c i u m , all the antibodies o f this class yield the same decoration pattern as the 'universal' anti-sequence a n t i b o d i e s D M I A and D M 1 B : t h e y d e c o r a t e all the m i c r o t u b u l a r n e t w o r k s e x c e p t for the cilia w h i c h are stained only at their tip under the fixation conditions used (fig 1 a, b). All these antibodies recognize only tx-tubulin on immu-
Fig 2. A, B. Immunoblotting analysis of Paramecium axonemes (A) and pig brain tubulin (B) with monoclonal antibodies and hybridoma supematants. The relative mobilities of cz- and fl-tubulias of both species visible on Coomassie blue stained gels (lanes CB in A and B) are inversed under the different gel conditions used (lower gel at pH 8.2 or 9.3). In A, 1.8/~g axonemes were loaded for Coomassie blue staining, and 0.2/.tg/well for immunoblotting. In B, 0.1/.tg of porcine brain tubulin were loaded in all wells, which is equivalent to the amount of Paramecium tubulin, assuming that axonemes contain 50% tubulin. Filters were incubated with antitubulin antibodies at dilutions listed below• Reference antibodies are quoted with * and monoclonals with o. Lanes 1-14: anti-sequence antibodies; 1, DM1A*: 1:1000; 2, DM1B*: 1:800; 3, TAP 9311: 1:100; 4, TAP 931B: 1:10; 5, TEU 435°: 1:100; 6, TEU 310°: 1:100; 7, TEU 441: 1:200; 8, TEU 4211G: 1:20; 9, TEU 4211H: 1:10; 10, TEU 4312: 1:10; 11, AXO 16: 1:104; 12, AXO 45°: 1:50; 13, TAP 926(3: 1:20; 14, TAP 956°: 1:100; lanes 15-22: other antibodies; 15, 6-11B-1": 1:100; 16, TEU 318°: 1:500; 17, TEU 348°: 1:250; 18, TAP 931E: 1:50; 19, AXO 49°: 1:3; 20, GT 335*: 1:250; 21, AXO 58: 1:2; 22, TAP 952°: 1:100. Revelation was performed with alkaline-phosphate secondary antibodies and NBT/BCIP. C. Dot-blotting analysis of antibody re.activities towards pig brain tubuiin in comparison with Paramecium axonemes. Line AXO Ix: 0.2/.tg of Paramecium axonemes/dot (corresponding to 0.1/zg tubulin); line PBT lx: 0.1/.tg of pig brain tubulin/dot; lines PBT 5x, 10x, 20x: 0.5, I, 2/.tg of pig brain tubulirddot. The filters were incubated with all the antibodies cited above, and some of them were chosen to illustrate the whole. The antibodies were more diluted than above because we performed an ECL revelation which is far more sensitive than the revelation with NBT/BCIP. Lanes: 3, TAP 9311: 1:200; 5, TEU 435°: 1:100; 6, TEU 310°: 1:100; 10, TEU 4312: 1:50; 11, AXO 16: 1:300; 12, AXO 450: 1:300; 13, TAP 926G: 1:20; 15, 6-11B-l*: 1:500; 16, TEU 318°: 1:800; 18, TAP 931E: 1:20; 19, AXO 490: 1:20; 20, GT 335*: 1:7,500; 21, AXO 58: 1:2; 22, TAP 952°: 1:100. Each antibody has been simultaneously tested on tubulin denatured in electrophoresis sample buffer (illustrated here), on native tubulin
Tubulin diversity in Paramecium
®
101
Paramecium axonemes (AXO) lml
/
, 9 ~ - < ~ ~-
.
123
Sle__
4
5
6
7
8
g
9 10 11 12 13 14 15 16 17 18 19 20 21 22
-" ,ta
..
°
II
antisequence
others
CB
"Pig brain tubulin (PBT) 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
(X
t
"
..
©
3
5
6
10
11
O O
O
O
12
AXOlx
O
PBT Ix
•
•
PBT 5x
O
•
•
*
PBT 10x
O
t
•
O
PBT 20x
O
•
o
O
13
O
15
0
16
18
O
0
19
20
O 'O
•
t"
•
O
O.
•
•
•
•
O
•
O
•
21
O
22
O
I
and on protein markers in sample buffer. The dot staining was identical or slightly more intense with native tubulin, and negative or very weak with unrelated proteins. To express the reactivity of the antibodies with pig brain tubulin relative to Paramecium tubulin, we indicate the ratio of the amounts of Paramecium tubulin and pig brain tubulin yielding a similar staining. For example: in (3) with TAP 9311, the reactivity has been noted 1 because pig brain and Paramecium tubulins were equally reactive; in (5) the reactivity of TEU 435 ° has been noted < 1:20 because at least a 20 times higher amount of tubulin from pig brain relative to Paramecium was necessary to give a similar staining. These reactivity values, determined in this way for all antibodies, have been indicated in table II (dot reactivity).
102
A M C a l l e n et al
noblots of (cx + ~)-tubulins from Paramecium and porcine brain (fig 2 A, B, lanes 1-11); however, they yield a more diffuse upward staining than observed with the reference antibody D M 1 A which takes the form of a thin, sharp band, suggesting that D M 1 A may be recognizing only a subset of ~-tubulin (see Discussion). To delineate the location o f the epitope recognized by these antibodies, they were each reacted on peptide maps of Paramecium a-tubulin as well as against various segments of ciliate ~x-tubulins obtained as fusion proteins (which allow to cover the complete c~-tubulin sequence of ciliates except for the N-terminal first 22 amino-acids, as shown in figure 3). Figure 4A shows, as expected, that: i) the reference antibody D M 1 A whose epitope is located between residues 426 and 430 of vertebrate c~-tubulin [11] does not react with Euplotes fusion protein (E) which terminates at residue 412, but reacts with the C-terminal fragment (C) o f Stylonychia fusion protein; ii) antibody TU-01, which recognizes an epitope located in the segment 65-97 o f vertebrate cx-tubulin [34], interacts with Euplotes (E) and Stylonychia N-terminal fN) fusion proteins. Similarly to TU-01, all the antibodies in this section react conspicuously with Euplotes (E) and Stylonychia N-t~rminal (N) fusion proteins at e q u i v a l e n t
FUSIONPROTEINS
25
Euplotes (E)
1
Stylonychia ft.
- -(N)
22
MW
(c)
176 214
PARAMECIUM A X O N E M A L u - T U B U L I N P E P T I D E S
3O
L . - ~
1
I
25 21 19 15 13 I1 7 6.5 6
k__i.
~
~Jl . . . I
I . . . . . .
J
I I .a L - - .m .Jm-t. t. _ .
.........
JI--HA.
. . . . .
J
J
k. _ _ . L , - - I , . . . . L - [ll,.i,.
_I
- _s
t.. . . . .
I
~
....
i
C
a-TUBULIN
N
I
3 5 4 4 6 5 97 |1
I
II
1
214 226
I
I
426 4 3 0 449 II I U DMIA"
I
I
6 - I 1 B - I * TU-01*
C98"
100 i
159 i
281 i
Ac
Fig 3. Schematic localization of the Paramecium axonemal a- and ~-tubulin antigenic determinants described in this paper. The diagram represents: I) the structure of fusion proteins corresponding to fragments of a-tubulin genes of the ciliates Euplotes (E) and Stylonychia (N, C); 2) Paramecium axonemal a- and ~-tubulin peptides generated after proteolysis by V8 protease and reactive with the antibodies studied. The molecular masses (Mr) of the peptides are indicated (in kDa). The peptide positions within the sequence are listed in table lII. Dotted lines represent the intervals between their'extreme possible positions. The epitope positions of reference antibodies (*) are those determined on brain tubulin and are represented on the whole polypeptidic chains of Paramecium or- and ~tubulins and on their fragments by solid rectangles (a white rectangle means that the concerned antibody has not been tested). The epitope positions of the collection of antibodies studied here are inferred from their re.activities on fusion proteins and on Paramecium tubulin peptides and include the uncertainties in the positions of the smallest reactive peptides. The names of antibodies are those of table II. Ac designates the three anti-acetylated tubulin antibodies, aS designates 8 anti-cx-tubulin sequence antibodies (except AXO 16) and flS I anti-fl-tubulin sequence antibody (AXO 45°). All the data recapitulated in this figure are illustrated in the paper, except for those concerning antibody C 140.
412
l
TAP 956 98
136
254
i
i
i
449 i
o.S
AXO$8, TAP 952
PARAMECIUM I3-TUBULIN P E P ' r I D E S
30
1 ............
~
......... i._
21 11 6
L . . . . .
~
t....
,.I.i
. . . . . . . .
Ill
....
J
i
13-TUBULIN
N I i
j
i
153 165 II
241 256 I
C 140"
C
416 430 442
I
II
C 113" 188 !
D
I B*
310 i
TAP 956
257
442
I
I
~S: A X O 4S 442
257 l
l AXO
58, T A P
952
Fig 4. Immunoblotting analysis on fusion proteins. Coomassie blue staining of a gel loaded with fusion proteins: Euplotes (E), Stylonychia: C-terminal fragment (C), N-terminal fragment (N) (see fig 3), and Paramecium axonemes (A). A. Reactivity of anti u-tubuhn sequence antibodies. Dilution of antibodies: DM1A*: 1:1000; TU-01*: 1:500; TAP 931B: 1:10; TEU 435°: 1:50; TEU 310°: I:100; TEU 4312: 1:20. B. Reactivity of anti-acetylated ¢x-tubulin antibodies. Antibody dilutions: 6-11B-I*: 1:100; TEU 318°: 1:1000. Revelation was carried out using alkaline phosphatase-labeled secondary antibodies and NBT/BCIP; ECL revelation gave the same results (not shown). In A and B, the amounts of fusion proteins loaded on the gels were roughly equivalent to that of Paramecium tubulin (0.2/zg of axonemes corresponding to 0.1 #g of tubulin) as determined by gel scanning. This is visible by comparison of the stainings yielded by the reference anti-vertebrate tubulin antibodies on fusion proteins and axonemes. C. Compared reaetivities on eukaryote tubulin (Tub E: Euplotes tubulin; Tub S: Stylonychia tubulin) and on fusion tubulin (E, C, N) of antisequence antibodies (*) and of antibodies negative in our standard conditions (in A and B) on fusion proteins. Antibody dilutions: TEU 435*: 1:500; DM1A*: 1:15,000; TAP 956°: 1:500; AXO 58: 1:4. ECL revelation was used in order to increase the sensitivity of detection. Protein
Tubulin diversity in Paramecium
Coomassie
DMIA*
103
TUOI*
TAP 931B
TEU 435
TEU 310
TEU 4312
ECNA
ECNA
E C
ECNA
E C NA
m
i
__..~
E CNA
..........
6-11B-l*
N A
TEU 318
Eupi Styi Para (E) (C) (N) (A) I
!
Fusion proteins
®
| °
,"
! E NA TEU 435*
DM1A*
©
E C N A
TEU 435*
TAP 956
t
~
AXO 58
~41
mn,
It i
o
TubE
l/ a' -l -~ TubS
E
-
TubS
C
.--.o
D
TubE
'N
TubS
E
TubE
C
N
TubS
E
C
N
quantities have been increased relative to those used in A and B: Tub E and Tub S x 3 (relative to P a r a m e c i u m tubulin); E x 3, C x 28, N x 5 so that the fusion protein amounts are at least equivalent to those of eukaryote tubulins. All antibodies strongly reacted with Tub E and Tub S; neither T A P 956 ° nor A X O 58 reacted with E and N; AXO 58 did not react with C either, whereas TAP 956* weakly reacted with C: compare the equivalent labelings provided by D M I A * and TAP 956 ° on Tub S, and the dramatically different stainings yielded by both antibodies on C.
104
AM Callen et al
MW V8
13
I 3
I
2
1
0
I MW 0
1
2
3
i - - 50
50_ t. 1
t
35--
_ 34 _ 30
30_
_ F
I
21--
1
O
11--
I,D,
,al~ll
,i~1~
I i~1~-
9
10
_ 25 -- 24
21 __19 11
6
I
2
3
4
5
6
7
8
11
Fig 5. Silver stained peptide map of Paramecium axonemal a- and fl-tubulins, a- and fl-tubulins were separated and cut out from gels of 10/.tg axonemes performed at pH 8.25; it is assumed that tubulin represents 50% of the axoneme protein content• Lane 1, Rainbow molecular mass markers of 46, 30, 21.5, 14.5, 6.5 and 3.5 kDa; in the standard gel conditions used here and in figures 6A and 12 (resolving gel at pH 8.25), the 3.5-kDa marker is visible at or under the bromophenol blue migration front. Lanes 2 and 11 (MW), molecular masses (in kDa) of fl- and a-tubulins and their major immunoreactive peptides derived from proteolysis by V8 protease, determined from their electrophoretic migration (see table III). Lanes 6, 7, 2.5/.tg of 13- or a-tubulins respectively, denoted 0 in the upper line 'V8'. Lanes 5, 4, 3 and 8, 9, 10, peptide generated by proteolysis of fl- or a- tubulin with respectively 0.002, 0.02 and 0.2/.tg V8, named 1, 2, 3 respectively in the upper line 'V8'. For all peptide maps, the same denomination will be used to indicate the protease amount. Compare the peptide pattern of fl-tubulin, spread in the MW range extending from 35 to 6 kDa, contrasting with that of a-tubulin which is clustered in 4 MW zones: 35-30, 20, 11 and 6 kDa. Note, in lanes 3 and 10, the presence of a 30-kDa component which is certainly 'V8' protease itself [19].
amounts relative to P a r a m e c i u m axonemal tubulin. As examples, results obtained with monoclonal antibodies and hybridoma supematants TAP 931B, TEU 435 °, TEU 310 ° and TEU 4312 are shown in figure 4A. Thus, the epitopes recognized by all these antibodies must be located in a sequence of a-tubulin comprised between amino-acids 22 and 176. The results obtained by immunoblotting of peptide maps fit very nicely with the broad localization deduced from blotting on fusion peptides and allow to narrow the domain in which the epitopes are located. Figure 5 shows peptide maps o f P a r a m e c i u m axonemal a - and fl-tubulins performed at three V8 protease concentrations. In spite of different patterns, the similarities in molecular masses of main peptides originating from a - and fl-tubulins suggest that they derive f r o m c l e a v a g e s which o c c u r r e d in similar regions of both polypeptidic chains; this is confirmed by immunoblotting analysis (see figs 3, 6, 12). As an example,
the peptide map staining pattern yielded by the monoclonal antibody TEU 435 ° is illustrated in figure 6A. This antibody strongly reacts with a-tubulin fragments of 34, 30, 25, 24, 21, 15 and 11 kDa and, more weakly, on fragments of 13 and 7 kDa (lanes 5-7). No fl-tubulin peptide is recognized, as expected (lanes 2-4). The other anti-a-tubulin sequence ( a S ) antibodies react with the same peptides (another illustration is given with TEU 310 ° in figure 10). To d e t e r m i n e the l o c a t i o n o f these peptides in the sequence of a-tubulin, a variety of reference antibodies were used. Key results were obtained with antibody TU-01, (directed to epitope 65-97) which recognizes all these peptides (fig 6A, lanes 8-11) including the 7-kDa one (hardly visible on lane 8), but not the 11-kDa one, which is hence located on the right (towards the C-terminal end) of residues 65-97. The positions of all these peptides are represented in figure 3 and table HI. Since all aS antibodies react with the 11 and 7-kDa peptides together, the epitopes they
Tubulin diversity in Paramecium
recognize must be situated in the overlapping region of both peptides, beginning approximately downstream residue 97 and limited by the C-terminal end of the 7-kDa peptide. The latter is not recognized by 6-11B-1 (see below) and hence covers the region 45 (or 65) to 102 (or 136). This delimits the region 98-136, indicated as ' a S ' epitope on the schematic diagram in figure 3 and in table II. All the results described so far could suggest that all these antibodies are identical. However, the immunofluorescence patterns obtained on metazoan cells, 3T3 and PtK2 (data not shown), differ depending on the antibody (see table II). TAP 9311 and TAP 931B decorate the whole microtubule network as DM1A and DM1B; TEU 435 °, TEU 441 give a varying labeling depending on the fixation mode used (formaldehyde after Triton X-100 extraction or methanol) as other antisequence antibodies do; the other antibodies decorate 3T3 and PtK2 microtubule arrays either weakly or not at all. These observations are confirmed by immunoblotting tests over pig brain tubulin (fig 2B, lanes 3-11): when using the same amount of this tubulin as that of Paramecium, antibodies TAP 9311, TAP 931B and AXO 16 are found to be clearly positive over the ct-tubulin subunit and in these cases, the staining is sharp and without any smear. The other antibodies react either much more weakly (TEU 435 °) or not at all (table II). To better estimate the extent of cross-reaction of these anti-ciliate tubulin antibodies towards mammalian tubulin, dot blots using increasing amounts of antigen were carried out (fig 2C and table II). The most strongly reactive antibodies are again TAP 9311 and TAP 931B; TEU 435 °, AXO 16 and TELl 4312 yield a weakly positive response; all the others, TEU 310 °, TEU 441, TEU 4211G and TEU 4211H are negative. In summary, all the antibodies in this class appear to be directed against sequence determinants located in the N-terminal portion, probably 98-136, of t~-tubulin. However, the epitope(s) recognized by TAP 9311 and TAP 931B is (are) common to ciliates and metazoa; the epitope(s) recognized by TEU 435 °, TEU 4312 and AXO 16 would be only partly conserved in metazoan tx-tubulin, whereas the epitope(s) r e c o g n i z e d by T E U 310 ° , T E U 4211G, T E U 4 2 1 1 H seem(s) to be ciliate-specific.
Anti-~-tubulin sequence ([~S) antibody Although this antibody was not tested on E coli expressed ciliate fl-tubulin, the strong similarity of its immunodecoration pattern with reference antisequence antibodies and, conversely, the distinction with those provided by antibodies recognizing post-translational modifications suggest that AXO 45 ° is directed towards a sequence determinant. Indeed, in immunofluorescence tests over Paramecium and metazoan 3T3 or PtK2 ceils (data not shown), AXO 45 ° yields a pattern similar to those provided by the universal antibodies DM1A and DM1B. In agreement with the immunocytochemical observations, AXO 45 ° reacts conspicuously on pig brain fl-tubulin both on immunoblots and dot blots (fig 2B, C, lane 12). On immunoblots of Paramecium axonemes,,antibody AXO 45 ° reacts only over the fl-tubulin, yielding a sharp band (fig 2A, lane 12). On peptide maps (fig 6A, lanes 11-17), this antibody reacts over a 21-kDa fragment of fl-tubulin, also recognized by the reference antibody DM1B thus indicating that this peptide, containing the flS epitope, comprises the C-terminal domain of fl-tubulin (fig 3, tables II and III). It is remarkable that this peptide is weakly stained by both antibodies, and especially by
105
AXO 45 °. No peptide resulting from c~-tubulin digestion is reactive. In conclusion, the epitope recognized by AXO 45 ° is located in the C-terminal domain of fl-tubulin and common to ciliates and metazoa.
Antibodies reacting weakly or not at all with fusion proteins The category now analyzed corresponds to antibodies that fail to react distinctly on fusion proteins. As an example, TEU 318 ° is shown in figure 4B; when gels are loaded with the amounts of fusion proteins on which the anti-tx-tubulin sequence antibodies reacted (fig 4A), this antibody is negative on tx-tubulin fusion proteins, either from Stylonychia (C, N) or from Euplotes (E). This result can be due to two main causes: either the antibody is directed against a posttranslationaUy added determinant not occurring in E coli, or the constraints on the potentially reactive tubulin segment are such that, when associated to fl-gaiactosidase, it is not accessible or not in the proper immunologically recognizable conformation.
Antibodies probably directed against or- and fl-tubulin sequences: TAP 956 ° and TAP 926G These two antibodies have the same characteristics. In immunofluorescence tests, they display the same pattern as DM1A, DM1B, and our anti-sequence antibodies both on Paramecium and on metazoan 3T3 and PtK2 ceils (table II). On immunoblots, they label both the tx and fl bands of Paramecium axonemal tubulin with approximately equal intensity (fig 2A, lanes 13, 14); they also recognize cytoplasmic tubulin but the tx band is more strongly labeled than the fl one (fig 7A). These results fit well with the fact that they decorate all microtubule networks in Paramecium. On peptide maps of fl-tubulin (fig 6B, lanes 2-4 and 8-11), the same peptides of 34, 30, 11 and 6 kDa are labeled by TAP 956 ° (table lll) and by the reference antibody C 113 [5] which recognizes an epitope located at the level of amino-acids 241-256 of fl-tubulin. On peptide maps of axonemal a-tubulin (fig 6B, lanes 5-8 and 12-14), TAP 956 ° strongly labels peptides of 34, 30, 25, 24 and 6 kDa (table III), and weakly labels a peptide of 5 kDa, exactly in the same way as the reference antibody C 98 [5] which is directed against an epitope localized in the central domain of tx-tubulin, as the level of amino-acids 214 to 226. The two extreme possible positions for the smallest (6 kDa) t - and tx-tubulin peptides recognized by TAP 956 ° can be deduced from their respective reactivities with C 113 and C 98: these two antibodies permit to delimit the regions 188-310 in t - and 159-281 in ct-tubulins including the epitopes recognized by TAP 956 ° and TAP 926G (fig 3 and table ID. In agreement with the labeling of metazoan cells, they cross-react with pig brain tubulin: on immunoblots, both label the tx-subunit and TAP 956 ° also labels the fl-subunit very weakly (fig 2B, lanes 13, 14); on dot blots, an amount of pig brain tubulin ten times higher than that of Paramecium is required to obtain reactions of similar intensity (fig 2C, lane 13). In immunofluorescence tests, TAP 956 ° and TAP 926G react on the microtubules of Stylonychia (not shown). They are also readily positive on immunoblots of the tubulin extracted from Stylonychia and Euplotes ceils (fig 4C, TAP 956 °, Tub S, Tub E). However, they did not react with amounts of fusion proteins equivalent to those of positively reacting Paramecium axonemal tubulin (as in fig 4]3); if they did not react either when the amounts of Euplotes
106
AM Callen et al •i.
®
T E U 435 I I
V8
13
I
2
3
TU-01*
I
oc
1
I
2
I 3
I
tl
30 21.5_
2
1
0
- t
ql~
O
FL.--
aid
~
4
(9
5
6
~,
•
3
I
2
1
1
1
~ 15
-11 - 7
7
8
9
10
11 12 13 14 15 C 113"
=
1.
3
-21-
-.
I
13
2
-25
~
I
I
I
-30
T A P 956
V8
I
l '-3
-
3
l
I
I 13 I eCl
g-
6.5_
2
I
P
i
4 Odlb
14.5 . . . .
I
IMWI
i ;
46
¢.
A X O 45
ec
3
tn
2
I 3
C 98*
I
MW
16 17
I
I
13 3
I
I
2
1
ct 1
2
I 3
r'T.
46 - -
30
0"
|a
-50-
21.5 -
!
14.5 - -
o g
m
Q
- - 25 - -
41.
,Q _11__
.o
~
O
_ 6 _ g o
!
6.5
~q
- 34--- 30--
T
--
0"
3.5--
1
2
3
4
Fig 6. Immunoblotting of peptide maps of
5
6
7
8
9
10
11
12
13
14
Paramecium axonemal ct- and fl-tubulins with anti-tubulin sequence antibodies, a- and fl-tubulins came from gels of axonemes carded out either at pH 9.25, in A (lanes 2-10) and in B or at pH 8.25, in A (lanes 12-17). Peptides arose from proteolysis of 3.75/Jg fl- or a-tubulin by V8 protease (see fig 5 for denomination of protease amount) except in A, lanes 16 and 17, where each track loaded with 5/.tg was divided in two halves after transfer. A and B, lane 1, rainbow coloured protein markers with their molecular masses; in B, in order to get a good resolution of small peptides (see for example, the 3.5 kDa marker), particular gel conditions were used, involving double concentrations of Tris at pH 8.8 (see Materials a n d methods); lanes 11 and 8 (MW),
107
Tubulin diversity in Paramecium
fusion protein were tripled (fig 4C, E), they nevertheless reacted w e a k l y when the amounts o f Stylonychia fusion protein (214-445) were multiplied by 28 (fig 4C, C). In conclusion, these data suggest that both antibodies are directed to a sequence epitope (see Discussion) present in the central domains o f ~z- and ~ t u b u l i n s (159-281 and 188-310, respectively), and common to ciliates and metazoa.
Antibodies certainly or presumably directed against posttranslational epitopes: TEU 318 °, TEU 348 °, TAP 931E, TAP 952 °, AXO 58, AXO 49 ° A large set o f antibodies was first assigned to this category on the basis o f i m m u n o c y t o c h e m i c a l tests: most o f these antibodies yield decoration patterns very similar to those obtained using reference antibodies directed against known p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s . T w o a d d i t i o n a l criteria were the absence o f immunoreactivity with E coli synthesized fusion peptides even at very high protein amounts (as an example, A X O 58 is shown in fig 4C) and, for one o f them ( A X O 49°), the delay in appearance o f immunoreactivity on assembled microtubules (see Discussion). These various antibodies could be subdivided into four clearly distinct groups, the first one directed against acetylated tubulin, and the last three assumed to be against other post-translational modifications.
Antibodies directed against acetylated ~x-tubulin (Ac antibodies). A s s i g n m e n t o f a n t i b o d i e s to this group h e a v i l y relied on the existence o f a very well characterized antibody directed against acetylated lysine 40 o f cz-tubulin, 6-11B-1 [44]. Three anti-Paramecium tubulin antibodies belong to this group: T E U 318 °, T E U 348 ° and T A P 931E, and their similarity with 6-11B-1 is striking in many respects. In immunofluorescence tests on Paramecium, they decorate cold or nocodazole stable microtubules and only the tip o f cilia (fig 8a) exactly as 6-11B-1 [2, 15, 70]. On immunoblots of Paramecium axonemal tubulin, they label only the cz-tubulin band, with a smear extending all the way to the position o f ~ t u b u l i n (fig 2A, lanes 15-18); in contrast to the strong staining yielded on axonemal tubulin, only a very small amount o f cytoplasmic tubulin migrating at the level
Table III. Estimated molecular masses and sequence positions of proteolytic fragments of Paramecium axonemai ~z- and ~tubulins reactive with the antibodies studied here. The molecular masses of the peptides were determined from plots of log M r vs migration after SDS-PAGE; mean values ± 2 standard deviations Listed below give a 95% confidence interval. The number of residues were calculated using 115 as mean molecular mass per residue. The peptide positions were inferred from their antigenic reactivities with reference antibodies, and the two extreme positions of their N- and C-terminal ends compatible with their reactivities were indicated, taking into account the experimental errors.
Apparent molecular mass (kDa) (daltons)
~x-tubulin peptides
,/)-tubulin peptides
34 30 25 24 21 19 15 13 11 7 6.5 6 34 30 21 11 6
33700 + 2200 30300 ± 2600 25100 ± 1400 23900 -+ 1400 20600_+ 1200 19200± 1200 14700 ± 2 800 12600 ± 3600 10700 ± 3000 6800 ± 1500 6500 ± 1100 6000 :e 1800 34200 ± 2200 2 9 7 0 0 ± 1800 20600 _+2000 11300±2000 6000±2000
Apparent number extreme of residues sequence positions N C 293 ± 13 263 ± 23 218 ± 12 208 ± 12 179 ± 10 167 ± 10 128 ± 24 110 ± 31 93 ± 26 59 ± 13 56 ± 10 52±13 297 ± 19 258 ± 16 179 + 17 98 ± 17 52 ± 17
1-35 1-35 1-35 7-35 1-35 254-293 1-65 1-35 98-147 45-65 1-35 159-214 1-138 1-153 257-281 166-241 188-241
274-346 240-320 226-264 226-254 169-213 430 ~ 9 104-213 102-175 164-213 102-136 46-100 226-281 278--415 256--415 430--442 256-355 256-310
o f the a x o n e m a l to-band reacts with antibodies 6-11B-1, T E U 318 ° and T E U 348 ° (fig 7B). These data validate the immunofluorescence ones showing a lack o f decoration o f c y t o p l a s m i c m i c r o t u b u l e s by these antibodies, h o w e v e r , .they do not account for the weak decoration o f the cilia.
molecular masses of cz- and ~-tubulins and their major immunoreactive peptides. In A, immunoblotting was carried out with TEU 435 ° (1:103) (lanes 2-7), TU-01* (1:5.103) (lanes 8-10), AXO 45 ° (1:2.5) (lanes 12-15), with respectively DMIB* (1:103) and AXO 45 ° (1:5) on each half-track of lane 16, and with respectively AXO 45 ° (1:5) and DM1A* (1:2.104) on each half-track of lane 17. It must be stressed that when ~tubulin bands were cut out from gels made at pH 9.25, in which/~-migrates faster than ot-tubulin, no staining of peptides was visible with anti-cz-tubulin sequence antibodies, even at long exposure times using chemiluminescence. In contrast, when ~tubulin was cut out from gels made at pH 8.25, in which the ~ b a n d is above the ~ one, we noticed a staining of peptides at the level of those deriving from a-tubulin. This can be attributed to contamination by ~z-tubulin on the primary gel, resulting from the slower migration of some forms of cz-tubulin which yield smeared bands extending all the way to the position of ~-tubulin on gels and blots (fig 2A, CB and lanes 3-11). Remark in lanes 16 and 17 the strong staining of the 19-kDa ~z-peptide afforded by DMIA* in comparison with the weaker staining of the 21-kDa/3-peptide yielded by DM1B*, used 20 times more concentrated. It seems that the lower amount of the latter peptide, visible on a silver stained gel (compare in fig 5 the staining of the 21-kDa ~peptide in lane 5 to that of the 19-kDa ~x-peptide in lane 8) cannot completely account for this difference. Additionally, with AXO 45 °, a still weaker staining of the ~peptide is exhibited (see Discussion), and no staining at all of the cz-peptide. In B, immunoreactivity with TAP 956 ° (1:10) in lanes 2-7, with C 113" (1/.tg/ml) in lanes 9-11, and with C 98* (0.6/ag/ml) in lanes 12-14. The same whole filter has been first treated with TAP 956* (monoclonai) then with a biotinylated anti-mouse IgG, and further processed as described in Materials and methods, yielding the staining observed on both ~z- and ~peptides in lanes 2-7; secondly, with C 98* (polyclonal) then with a biotinylated anti-rabbit IgG, producing the staining of a-peptides only, seen in lanes 12-14; thirdly, with C 113" (polyclonai), resulting in the labeling of/J-peptides solely, visible in lanes 9-11. The same results had been obtained after single staining of ~z- and ]3-peptides with the three antibodies, and in particular, C 98* did not label/~-peptides whereas C 113" did not stain the cr ones. Note in lane 6, the presence of a peptide stained by TAP 956 ° at the level of the/~ 11-kDa peptide and not labeled by C 98*; it therefore appears to result from a contamination of ~z- by ~tubulin since the tubulin bands were cut from gels of axonemes made at pH 9.25, in which some forms of ~-tubulin migrate slower and are spread until the level of ~z-tubulin. Conversely, this staining is not seen when the tubulin bands are cut from gels made at pH 8.25 where (x-migrates faster than/3-tubulin.
108
A M C a l l e n et al
I
, ct ,
4
,i
i
i
" "! i=-la la -
II -
[ i [
Axo Cyt
Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt I II II I
A
B
C
Anti-acetylated tubulin antibodies Fig 7. Comparative immunoblotting analysis of Paramecium axonemes (Axo) and cytoplasmic (Cyt) extract. Relative reactivities of antibodies which label weakly (A) or not at all (B, C) fusion proteins. Protein mounts: axonemes, 0.3 gg; the cytoplasmic extract contained an equivalent amount of tubulin (see the relative staining of DM1A* on Axo and Cyt). ECL revelation was carded out. A. Presumed anti-tubulin sequence antibodies: TAP 926G: 1:500; TAP 956°: 1:103. Both ix- and fl-tubulins are labeled. B. Anti-acetylated tubulin antibodies: 6liB-l*: 1:500, TEU 318°: 1:104, TELl 348°: 1:2.10 3, TAP 931E: 1:600. Only tx-tubulin is stained, but very weakly in cytoplasmic extract` C. Antibodies directed against putative post-translational modifications different from acetylation: GT 335*: 1:105, AXO 58:1:103, TAP 952°: 1:4.104. GT 335* reveals on axonemes a doublet the faster component of which migrates like fl-tubulin and is also stained by AXO 58 (which reacts with additional bands); on the cytoplasmic extract, reactivity is seen at the level of fl-tubulin, as with AXO 58; high moleeul~ mass polypeptides are also visible, as noted by Br6 et al [10]. TAP 952 ° labels both fl- and tx-tubulins.
On peptide maps of t~-tubulin, the four Ac antibodies strongly react over the same N-terminal peptides of 34, 30, 25, 24 and 21 kDa and faintly on lower molecular mass peptides (fig 3; fig 9, lanes 3-9; table IT/). No fl peptide is stained (fig 9, lanes 1-2). In order to narrow the localization of the epitopes recognized by these Ac antibodies, a comparison of their reactivities with those of the anti-ct-tubulin sequence (tzS) ones was carried out focussing on the low molecular mass peptides (fig 10). The smallest peptide well recognized by both categories of antibodies is a 13-kDa one, whereas the smallest peptide reactive with all Ac antibodies is one of 6.5 kDa. The latter peptide differs from the 7-kDa one reactive with the aS antibodies; this 6.5 kDa peptide permits to delimit the region 1-100, indicated as 'Ac' epitope on the diagram in figure 3 (see also tables II
and 1II). As 6-11B-l, the Ae antibodies are fully negative on all types of tz-tubulin fusion proteins (as an example, TEU 318 ° is shown in figure 4B) even if amounts of proteins are very high. Immunoblotting tests over pig brain tubulin yield a sharply decorated band, without smear, of variable intensity with the different antibodies (fig 2B, lanes 15-18). This is confirmed by dot blots: TAP 931E reacts on pig brain tubu-
lin only when a 10 times higher amount is deposited with respect to Paramecium axonemal tubulin; with TEU 318 ° and TEU 348 °, a five-fold ratio is sufficient to obtain equivalent intensity while 6-11B-1 is intermediate (fig 2C, lanes 15, 16, 18). As 6-11B-1, the three antibodies fail to decorate microtubules in PtK2 cells, the tubulin of which is non-aeetylated [54] while they decorate the stable microtubule network in 3T3 cells (not shown). In the latter cells, the primary cilium, the midbody and the mitotic spindle are also strongly decorated. To definitely establish that our antibodies are indeed directed against acetylated tz-tubulin, we performed chemical acetylation on native purified pig brain tubulin. As already observed by Piperno and Fuller [53], acetylated tubulins, o~ Ac and fl Ac, migrate slower than their native counterparts, tz and fl (fig 11A: Coomassie blue and blots with DM1A and DMIB). This slower migration is very likely due to decreased SDS binding resulting from neutralization of basic lysine residues [16]. It can serve as a 'signature' of tubulin acetylation. Note that native porcine brain tubulin contains a minor amount of acetylated tubulin migrating slighty higher than the major native t~-tubulin revealed by DM1A (fig l l B ) , whereas a strong reaction with the four antibodies is visible on in vitro fully acety-
Tubulin diversity in Paramecium
109
:,
b ~''~
',' /
.~
,,
i','~ /
'
.~ \,
Fig 8. Projections of Paramecium cells decorated with the following antibodies: (a) TEU 348 ° (1:10), (b) TAP 952 ° (1:20), (e) AXO 58 (1:2) and (d) AXO 49 ° (1:5) observed by confocahmicroscopy. Each antibody yields a specific staining of the cilia: only the tips (arrowheads in a) are strongly decorated with TEU 348 °, while, in addition, the rest of the axonemal structure is decorated along its whole length with TAP 952 ° (b) or only along its proximal part with AXO 58 (c) including the tips (arrowheads in b and e); in contrast, the strong decoration of axonemes by AXO 49 ° (d) contrasts with that yielded by the other antibodies, a. This projection collecting all the information from inside the cell shows that only some microtubules are decorated with the TEU 348° antibody: subpellicular microtubules associated with the anterior left area of the cell (P), postoral fiber (Po) and cytopharyngeal microtubules (Cf) associated with the oral apparatus (O), microtubules associated with the contractile vacuole system (V). In contrast, the TAP 952* antibody Co) also decorates the internal meshwork of microtubules (arrows). AXO 58 (e) yields a pattern similar to TEU 348 °, while AXO 49 ° (d) is more restrictive: it decorates only the postoral fiber (Po) associated with the oral area (O) and a short portion of the microtubules associated with the contractile vacuole system ((V), partly out of the focus). Scales are 254//m.
1 10
AM Callen
T E U 318
I I
I
V8
3
6-11B-I*
I I
a
1
1
et al
2
3
I I
a 3
2
MW
1
'
MW -30 -25
R
~ ~
-45 -34
-21
30 ~
-15
-13 -11
- 25 -21
-7
--10
1
2
3
4
5
678
9
Fig 9. Immunoblotting of peptide maps of Paramecium axonemal a- and fl-tubulins with anti-acetylated tubulin antibodies. Here, tubulins were separated from a gel of axonemes at pH 9.25 in order to avoid contamination of 13- by a-tubulin. Peptides arose from digestion of 3.75/Jg fl- or cx-tubulin by V8 protease (see figure 5 for protease amounts). Immunoreactivity with TEU 318 ° (1:200) (lanes 1-5) and with 6-11B-I* (1:50) (lanes 6-8). Lane 9 (MW), molecular masses (in kDa) of major immunoreactive peptides.
lated a-tubulin, together with a large migration shift. N o reaction is detectable on fl-tubulin in all cases (best illustrated with 6-11 B- 1 and T A P 931E in fig 11B). In conclusion, these three new anti-tubulin antibodies are clearly directed against an N-terminal acetylated epitope o f a-tubulin. T A P 9 5 2 °. T h i s a n t i b o d y a t t r a c t e d a t t e n t i o n b e c a u s e it a p p e a r e d to d e c o r a t e both the c i l i a along their w h o l e lenght and nearly all the i n t r a c e l l u l a r m i c r o t u b u l e n e t w o r k s o f P a r a m e c i u m (fig 8b). W h e n T A P 952 ° is c o n s i d e r a b l y diluted, only the tip o f cilia is l a b e l e d and w h e n l e s s d i l u t e d , a g r a d i e n t o f l a b e l i n g is o b s e r v e d d e c r e a s i n g f r o m the p r o x i m a l to the d i s t a l p o r t i o n o f cilia, as has been observed, albeit less clearly, with A X O
Antibody
58 (fig 8c). On i m m u n o b l o t s obtained from gels m a d e at pH 8.2,
1 2 Fig 10. Compared immunoblotting of a peptide map of Paramecium a-tubulin with an anti-acetylated tubulin antibody (lane 1) and an anti-a-tubulin sequence antibody (lane 2). Peptides were generated by proteolysis of 5 pg o~-tubulin by 0.2/.tg V8 protease and separated in standard gel conditions at pH 8.25; after transfer, one track was cut in two halves; one half-track (1) was incubated with 6-11B-l* ascitic fluid (1:103) and the other (2) with TEU 310 ° (1:200). Note the good visualization of minor peptides achieved here (in contrast to fig 9) by using high protein loadings and high antibody concentrations (6-11B-1" ascitic fluid instead of culture supernatant) and the resolution of the 7 and 6.5 kDa bands. Our anti-acetylated tubulin antibodies stained the same peptides as 6-11B-I*.
T A P 952 ° reacts with both o~- and fl-axonemal tubulins. The bands are sharp and without smear (fig 2A, lane 22). The antibody also reacts with P a r a m e c i u m cytoplasmic tubulin, the staining at the level o f fl being stronger than at the level o f o~-tubulin (fig 7 C). These results fit well with the labeling of both cytoplasmic and axonemal microtubules observed by immunofluorescence. On i m m u n o b l o t s o f p e p t i d e m a p s , T A P 952 ° r e a c t s with axonemal peptides o f 21 k D a from fl-tubulin and o f 19 k D a f r o m ~ - t u b u l i n , a l s o l a b e l e d b y D M 1 B a n d D M 1 A , respectively (fig 12, lanes 11-20), and therefore situated in the C-terminal d o m a i n o f each subunit (fig 3 and tables II, III).
Fig 11. Demonstration of anti-acetylated a-tubulin antibodies by means of in vitro acetylation of pig brain tubulin. Long gels (12 cm) were used to resolve acetylated co- and ]J-tubulin bands. Tubulin amounts: Coomassie blue: 0.8 pg/well before (C) and after acetylation (Ac); blots: 0.4 pg/well. Antibody .dilutions: DMIA*: 1:5.104, DM1B*: 1:5.104, 6-11B-I*: 1:5.102, TEU 318°: 1:4.104, TAP 931E: 1:50, TEU 348°: 1:5.103. ECL revelation was carried out. A. Coomassie blue: note the slower migration rates of a- and fl-tubulins after acetylation: compare a Ac with ~, fl Ac with ft. DM1A* and DMIB* label respectively ct- and fl-tubulin before (C) and after acetylation (Ac); migration shifts are visible on blots as seen on the gels. The level of the bands stained by DMIA* is slightly higher than the positions of the other bands because they correspond to tracks situated on the left side of the gel. 6-11B-l*, TEU 318 °, TAP 931E, TEU 348 ° label strongly a-tubulin after acetylation whereas they yielded a very weak staining on native pig brain tubulin. B. In order to get a
Tubulin diversity in Paramecium
®
l 11
Coomassie Blue
Or,--
•
B
0¢ Ac -Ac
"
-
,
-.
m
,q
f--
-m
.me
.-,D
C Ac
C Ac
C Ac
-
Ac
C
C Ac
,
®
C Ac
,
.
C Ac
j
¢xAc Gt
d - - 13Ac
I
II
before acftylation (C)
after acetylation (Ac)
better separation of ¢x- and ~-tubulins, SDS from Sigma has replaced SDS from BDH [8] in the gel and electrophoresis buffers. After transfer, each track has been cut in two halves and treated by two different antibodies. Before acetylation, 6-11B-1" labels weakly a minor form of a-tubulin not stained by DMIA* and which migrates slightly slower than the major form labeled by DM1A*. After acetylation, 6-11B-I* and TAP 931E label strongly a thick band at the level of that stained by DMIA* (c~ Ac) and faintly a band situated lower, also visible in A. The labeling of the acetylated ]3-tubulin by DMIB* (~1Ac) at a still lower level shows unambiguously that the faint band does not correspond to acetylated ~-tubulin.
112
AM Callen et al
AXO 58
I I
II
1
II
I
ll31O IMwl
I 2
V8
TAP 952
.<
0
0
1
2
3
1
1
1
0
~.
II
I
13 2
~
I
I 131 0
1
2
3
1 IW
30
1 r
•
OQ
46_
I
-
21.5-
°
~'
,21, "=- 19-
0,~
QB,
°
='QD
14.5 6.5_
1
2
3
4
5
6
7
8
9 10
11 12 13 14 15 16 17 18 19 20
Fig 12. Immunoblotting of peptide maps of Paramecium axonemal ¢x- and ~tubulins with presumed anti-tubulin post-translational modification antibodies. Here, tubulins were separated from a gel of axonemes at pH 8.25. Peptides were yielded from proteolysis by V8 protease of 3.75 #g/3- or ¢x-tubulin (lanes 2-3, 6-8), 5/Jg (lane 9), 2.5 #g (lanes 10, 12-13, 16-20). Undigested/3- and a-tubulin amounts were 3.75/tg (lanes 4-5) and 1/.tg (lanes 14-15). In lanes 9, 10, 19, 20, each track was cut after transfer in two half-tracks. Lane 1, protein markers and their molecular masses (in kDa). 11 (MW), molecular masses of undigested a- and ~-tubulins and their immunoreactive peptides. Immunoreactivity with AXO 58 (undiluted) in lanes 2-8 and on half-tracks of lanes 9 and 10; TAP 952 ° (1:20) in lanes 12-18 and (1:50) on half-tracks of lanes 19 and 20; DMIB* (1:103) and (1:5.103) on half-tracks of lanes 9 and 19, respectively; DM1A* (1:2.104) and (1:104) on half-tracks of lanes 10 and 20, respectively. Remark in lanes 9 and 10 that the equivalent staining of the 21-kDa ~]-peptide with DM1B* and DM1A* on one hand, and with AXO 58 on the other hand, is due to the different quantities o f ~ and a-tubulins (5/zg versus 2.5/zg). When an equivalent low tubulin amount (2.5/Jg) is loaded as in lanes 19 and 20, the labeling of the 21-kDa/~-peptide with DM1B* and AXO 58 is very faint. Note also the presence of an additional fl-peptide of ~ 19 kDa, labeled by DM1B* and only visible at high protein levels (compare lane 9 here and 16 of figure 6A with lane 19 of this figure). Note in lanes 19 and 20 the stronger staining of the 19-kDa ~x-peptide compared to that of the 21-kDa/ff-peptide by TAP 952 °, which could be probably correlated with the lower amount of the latter peptide.
T A P 952 ° does not react with fusion proteins (not shown). It fails to react with pig brain tubulin in immunoblotting and with c y t o p l a s m i c m a m m a l i a n tubulins in immunofluorescence tests (fig 2B, C, lane 22 and table II). This antibody therefore appears to be ciliate-specific, at least as concerns the cytoplasmic microtubules (note however that the cilia of metazoans have not been tested). A X O 58, an antibody yielding a pattern similar to G T 335, an anti-glutamylated tubulin antibody. A X O 58 was not successfially sub-cloned. However, we present some of its characteristics which are unique while others are reminiscent of GT 335 antibody which is known to react with polyglutamate side chains located in the C-terminal part of tubulin [75]. In immunofluorescence tests on Paramecium cells, AXO 58 decorates cilia in both the proximal and distal domains, as well as a stable population of cortical and cytoplasmic microtubules (fig 8c). This is very similar but not identical to the pattern provided by antibody GT 335 which, in addition, labels a sub-population of dynamic mierotubules [10].
On immunoblots obtained from gels of Paramecium axonemes made at pH 8.2 (fig 2A, lanes 20, 21), AXO 58 labels a band which migrates at the level of ~-tubulin, as was observed with GT 335, but also a band at the level of a-tubulin not recognized by GT 335 (fig 7C). Conversely, GT 335 stains a band which migrates just above ~tubulin, apparently not recognized by AXO 58. Thus, both antibodies seem to react with different forms of axonemal tubulin, which display different apparent molecular masses on SDS gels. Finally, as GT 335, AXO 58 reacts with cytoplasmic tubulin, at the level of the fl subunit (fig 7C). On immunoblots of peptide maps, AXO 58 mainly reacts with 21-kDa and 19-kDa peptides respectively originating from axonemal ~ and ¢x-tubulins, and respectively also labeled by D M I B and DM1A (fig 12, lanes 2-11), indicating that they are both located in the C-terminal part of each subunit (fig 3 and tables II, IlI). This result is somewhat different from that obtained with GT 335, which labeled, after tubulin proteolysis, peptides displaying shifts in migration relative to those of the 21- and 19-kDa ~- and o~-peptides
Tubulin diversity in Paramecium
!f
1 13
\
,j
I:1
,
Fig 13. Two projections of Paramecium cells at approximately the same stage of division and decorated with (a) TEU 348 ° (1:10) and (b) AXO 49 ° (1:5) antibodies. In addition to the oral apparatus (O) and the cilia, only microtubular rootlets associated with the two contractile vacuole systems (V) are decorated by AXO 49 ° (b). The latter correspond to parental systems (Vp), decorated by both antibodies (a, b), while the newly assembled ones (Vn in a) are not decorated by AXO 49 ° (b). In addition, transient structures, such as the elongation spindles of the micronuclei (e) or the microtubules located inside the macronucleus (ma), visible in (a) are again not decorated by AXO 49 ° (b). Scales are 25 #m.
(Levilliers et al, in preparation), again suggesting that different forms of tubulins would be recognized by both antibodies. Since/3-tubulin fusion proteins were not available, only the test against cx-tubulin fusion peptides could be carried out and proved to be fully negative, even when using high levels of fusion peptides, containing amounts of tubulin at least equivalent to that of the corresponding eukaryote tubulins on which the AXO 58 antibody reacts (in fig 4C, A X O 58, compare E, C, N to Tub E, Tub S). AXO 58 fails to react both on microtubules of PtK2 and 3T3 cells in immunofluorescence tests (table II) and on pig brain tubulin in immunoblots and dot blots (fig 2B, C, lane 21). This strongly contrasts with the distinct reactivity of GT 335 on PtK2 and 3T3 cells (not shown) and on pig brain tubulin both in immunoblots and dot blots (fig 2 B, C, lane 20).
AXO 49 °, an antibody directed against a post-translational modification similar to that recognized by a polyclonal antibody. This antibody is one of the few monoclonals which decorates strongly, in Paramecium, the cilia along all their length, except their tip, along with a restricted subpopulation of very stable microtubules (fig 8d). Its immunocytochemical labeling pattern is very similar to that yielded by a rabbit polyclonal serum raised against axonemal tubulin of Paramecium [14]. In addition, as in the case of the polyclonal serum [2] the reactivity of A X O 49 ° is detected over microtubules only
following a substantial lag after their assembly. For example, figure 13 shows that the newly assembled contractile vacuole system is not labeled by A X O 49 °, while it is decorated by TEU 348 °, an anti-acetylated tubulin antibody. On immunoblots of Paramecium axonemes, A X O 49 ° reacts with both ~x- and ]~-axonemal tubulins (fig 2A, lane 19). In contrast, it is fully negative over cytoplasmic Paramecium tubulin (not shown), in agreement with the immunocytochemical data. Moreover, this antibody is fully negative on cytoplasmic metazoan tubulin as shown by immunofluorescence tests over 3T3 and PtK2 cells (table II) as well as on immunoblots and dot blots over pig brain tubulin (fig 2B, C, lane 19). These data, combined with the fact that A X O 49 ° does not react over fusion proteins, strongly suggest that this antibody recognizes a post-translational epitope; it will be characterized in detail in a forthcoming paper (Levilliers et al, in preparation).
Discussion At the start of the present work, we had set ourselves the aim of isolating an array of monoclonal antibodies to ciliate tubulin as broad and diverse as possible. This is why we introduced variations in the type and mode of presentation of the tubulin antigen. Out of three fusions, a large diversity of antibodies was obtained, including both sequence and
114
AM Callen et al
post-translational modification-directed ones and, in particular, among the latter, a set of four antibodies, three of which correspond to new specificities previously undescribed in the literature. We will analyze the strength of these conclusions, evaluating the level of certainty with which both the nature and the location of epitopes has been established.
cut from the same track (see fig 6A, lane 16, fig I0 and fig 12, lanes 9-10, 19-20). This can be confirmed by double staining with both antibodies but one cannot exclude a comigrafion of two different peptides which would account for the simultaneous staining of a band by two antibodies. The well identified anti-o~-tubulin sequence antibodies and the location of their epitope(s)
Methodological considerations on peptide maps
The location of the epitopes has been determined through immunoblotting of peptide maps generated by various concentrations of V8 protease, yielding an array of peptides ranging from large molecular masses (30-20 kDa) to small ones (5 kDa). One preliminary point related to the structure of the protein under digestion and several comments on the technical advantages and some pitfalls of the method used can be made. The cleavage sites generated by V8 protease on SDSmercaptoethanol-treated Paramecium tubulin in acrylamide we inferred from the peptide sizes are situated in regions 100-200 and ~250--300 of ~x- and/$-tubulins also found by Serrano et al [63] and De la Vina et al [17] after cleavage of native soluble bovine brain tubulin by various proteases. This suggests that the so-called 'denatured' tubulin such as used in the 'Cleveland' procedure would not be completely unfolded and would keep a certain degree of its initial three-dimensional structure as compatible with 3 proteolytitally defined large compact regions [17] consisting of the N-terminal, middle and C-terminal thirds of the molecule: these regions presumably correspond to real structural features of native tubulin. The simultaneous separation of high and low molecular mass peptides on a single gel was rendered possible by the use of a 15-20% acrylamide gradient, which is not common for peptide map blotting. For most peptide maps presented here, involving a lower gel at pH 8.25, peptides of 6.5 kDa were well resolved (see figs 6A and 10), whereas, by using double "Iris concentrations both in the lower gel buffer (adjusted at pH 8.8) and the running buffer, peptides of 3.5 kDa could be resolved (compare the M r markers of fig 6A and B). Finally, the use of ECL revelation for immunoblots permired to visualize peptide staining otherwise not detected by classical coloured revelations (using NBT/BCIP or diaminobenzidine). This was the case for the small peptides of 11 and 7 kDa recognized by the anti
When a monoclonal antibody remains reactive over a portion of tubulin synthesized in E coli, we conclude that the antibody is directed to a sequential epitope of tubulin. This is corroborated by two 'internal controls': reference antibodies known to be directed to a primary sequence determinant such as TU-01 or DM1A indeed remain positive over fusion proteins and, conversely, 6-11B-1, an antibody whose reactivity is known to depend on the post-translational acetylation of lysine 40 [44] indeed completely fails to react over fusion proteins. In addition, all our antibodies that are positive on fusion proteins yield on Paramecium cells immunoeytochemical decoration patterns similar to those of reference anti-sequence antibodies and quite different from those of reference anti-modification ones. On the basis of these arguments, nine of our antibodies have been classified as being directed against an a-tubulin amino-acid sequence in the broad sense, the contiguity of which would result either from the primary structure or from the polypeptidic chain folding. The location of the epitope(s) has been further narrowed by peptide map blotting. For eight of the anti-cz-sequence (o~S) antibodies, the epitope appears to be located between positions 98 and 136 as deduced from the two smallest peptides (of 11 and 7 kDa) which they recognize (fig 3 and tables II, 131); however, taking in account the uncertainties in molecular mass and peptide position determinations discussed above leads to a conservative estimate of 150 for the extremity of the 7-kDa peptide. The region 98-150 of Paramecium cz-tubulin therefore appears to be specially immunogenic, in agreement with predictions of antigenic determinants of brain tubulin based on amino-acid hydrophilicity and with the fact that it corresponds to a zone of preferential cleavage by proteases [17, 49], hence probably accessible and immunogenic. In fact, it is also in the region 90-160 that Wolff et al [74] locate the epitope of the monoclonal antibody 111 B52 C2 directed against pig brain cx-tubulin. Moreover, this antibody seems to label the same peptides of Paramecium axonemal tubulin as our anti-sequence antibodies (not shown). In fact, our eight antibodies are not identical: one (TAP 931B) belongs to the IgG3 class while all the others belong to the IgG1 one; moreover, their reactivities against pig brain tubulin differ. We have tried to identify amino-acid substitutions distinguishing ciliates from mammals: they could.be immunogenic when ciliate tubulin is injected into a mouse and could belong to the epitopes of the ciliate-specific aS antibodies, ie at least TEU 310 °, TEU 4211G, TEU 4211H. When conservative substitutions are discarded, five amino-acid differences shared by all ciliate cx-tubulin sequences examined (12 belonging to nine species) are identified in the region 98-150: Cys/Val at position 118, AsrdGln at position 128, Val/Phe at position 141, Leu/Phe at 149 followed by Gly/Thr at position 150 (fig 14A). As, all the antibodies of this category are strictly cz-tubulin specific, it may be inferred that the epitope(s) towards which they are directed is (are) absent in the ~chain. Comparing sequences of both ~- and/~-polypeptidic chains of Paramecium tubulin in this region (fig 14B), it can be seen that they are identical
Tubulin diversity in Paramecium
Parameczum
91 QLISGKEDAA
Mammal
---t ........
Paramecium ct M~--.~ 1
Paramecium
NNFARGHYTI y .......
RIRKLADNCT
GLQGFLVFHS
Q
VGGGTGSGLG F . . . . . . . FT
151 Sr.7.7.~.RLSVD Y G K K S K L G F T I Y P S P Q V S T A .m. ~--S - - - A
VVEPYNSILS THSLLEHTDV t --tT---s-C
CVMLDNEAIY A F - v ......
211 DICRRNLDIE
SLRFDGALNV
270 PYPRIHFMLC .P-A
RPTYTNLNRL
Mamm~ i
®
GKEIVDLCLD .... i - - V - -
115
IAQVISSLTA S-iv--i
91
DITEFQTNLV 1
4,4,
4,
Paramecium QLISGKEDAA Paramecium NFvF-QTG-G
NNFABEHYTI GKEIVDLCLD --W-k .... E - A - I i - S V - -
RIRKLADNCT Vv--E-eG-D
89
Paramecium
151 ST.T.T.r.RL~. Y G K K S K L G F T
P a , r a m e e i mn t - - i S k v R E e - P D r I M E T - s
IYPSPQVSTA
I S I L 5
iV---K--DT
~ k T - -
IA0VISSLTA vSAAm-Gv-C
SLRFDGALNV C---~-Q--S
THSI~HTDV V-Q-v-NA-E
149
211 Paramecium ~_T_~E Paramecium
270
RPTYTNL~ T T---GD--H-
209
268
Fig 14. Partial amino-acid sequence comparison of tubulins. A. Paramecium and mammalian consensus a-tubulin sequences. The arrows point the positions of non-conservative substitutions between residues 98 and 150. The lower-case letters indicate the conservative substitutions. B. a- and ~tubulins from Paramecium. A gap was introduced in ~tubulin in order to obtain the best alignment. The numbering of residues is therefore different between a- and ~- tubulins. Negative types indicate homologous regions between a- and ~- tubulins at the potentially immunogenic sites (see Discussion). The homologous regions u(272-277), ~(270-275) have not been displayed because they contain one non-conservative substitution. Paramecium a- and ~-tubulin sequences are from P Dupuis [20, 21]; mammalian u-tubulin consensus sequence (from 14 sequences belonging to five mammalian species) and alignments were compiled by H Philippe (personal communication).
from residues 138 to 150, except at two positions where conservative substitutions are found: at 141, Val/Leu and at 149, Leu/Met; the sites at positions 141, 149 and 150 are thus excluded. At positions 118 and 128, amino-acid differences are found between the a and fl chains: Cys/Val including a polarity change and Asn/Gly without any polarity change; they remain as potential candidates for the antigenic determinants of the ciliate-specific o~ antibodies. Finally, it can be r e m a r k e d that the a n t i - a - t u b u l i n sequence (aS) antibodies yield a spread staining on Paramecium axonemal ~x-tubulin, extending all the way to the level of the ~-subunit, as the anti-acetylated tubulin antibodies do (fig 2A). In contrast, on Paramecium cytoplasmic and pig brain ~-tubulins, these antibodies labeled fine bands (figs 7B and 213). This suggests that the c~S antibodies recognize, in axonemal tubulin, different a-tubulin isoforms resulting from various axonemal-specific post-transl a t i o n a l m o d i f i c a t i o n s that w o u l d be c a u s i n g slight differences in eleetrophoretic migrations (see for example, the shifts of migration of acetylated pig brain tubulin, in fig 11). The lack (or weakness) of reaction of the smear on Paramecium axonemes with other anti-sequence antibodies
such as D M I A and D M I B could be attributed to their insufficient affinity for the minor isoforms which are detected by Coomassie blue only on highly loaded gels. In conclusion, although some uncertainty remains as to the precise amino-acids involved, the location of the epitopes recognized by our anti-sequence antibodies has been defined within a relatively short peptide domain. The A X O 45 ° anti-~-tubulin antibody: a presumptive antisequence probe
In the absence of ~-tubulin fusion proteins, the discrimination between anti-fl sequence and anti-fl modification antibodies could only be based on immunocytochemical arguments. T h e r e f o r e the d e s i g n a t i o n of A X O 45 ° as an anti-sequence antibody was based on the similarity between its labeling pattern and that of reference anti-sequence antibodies, such as DM1A and DM1B. The epitope has been localized on a 21-kDa peptide starting at residue 257, also recognized by DM1B, ie situated in the C-terminal domain of the ~-tubulin molecule (fig 3). Since the reactivity of AXO 45 °, as of DM1B, is
116
AM Callen et al
much weaker on this peptide than on the whole molecule, it can be inferred that both antibodies recognize conformational determinants in addition to a sequence one. This agrees with the respective observations of Breitling and Littie [11] and of De la Vina et al [17] on the decrease of the reactivity of DM1B on fl-tubulin peptides and on a synthetic peptide 13 (412--431) in comparison with that on whole tubnlin. The case of anti-sequence antibodies that only react very weakly on fusion proteins
Two antibodies TAP 956 ° and TAP 926G, both directed against tx and fl-tubulins, yield immunocytochemical decoration patterns on Paramecium and metazoan cells very similar to those of the bona fide anti-sequence antibodies just discussed; but, in contrast to the latter, they react only with very high amounts of Stylonychia fusion protein (21A. A,d5). This weak response appears to be significant for several reasons. First, increasing the amounts of fusion protein loaded on a gel in the same proportions never yielded positive results with antibodies known with certainty to be directed against post-translational modifications (see below). Second, the central location in Paramecium ct- and fl-tubulins of the smallest peptides reactive with TAP 956 o and TAP 926G, at positions 159-281, and 188-310, respectively (fig 3) in a region where no post-translational modification has yet been found, suggests that the epitopes recognized by these antibodies correspond to sequence determinants. Third, at least some parts of this region are immunogenic, as evidenced by: i) the isolation of the antibodies C 98 and C 113 from synthetic peptides t~ (214-226) and fl (241-256) chosen from theoretical antigenlcity predictions [17]; and ii) the obtention, using microtubules or cleaved tubulin as immunogen, of antibodies directed to the central domains of both tx- and fl-tubulins [30, 74]. Because the two antibodies react both on Paramecium ani] mammalian ix- and fl-tubulins, we searched aligned sequences of both tx- and fl-tubulins in the 160o310 domain for strongly similar amino-acid stretches that might constitute the epitope (fig 14B). Three such stretches were found: tz(180-186) and fl(178-184), a(201-213) and fl(199-211), and tx(258-267) and fl(256-265). Comparison with the stretches against which De la Vina et al [17] generated their anti-synthetic peptide antibodies is quite interesting. The antibodies they obtained, directed towards the peptides a(214-226) and/3(241-256), contrary to ours; turned out to be subunit-specific. A third antibody, C 140, directed to fl(153-165), was not reactive with the 6-kDa Paramecium fl-peptide (fig 3). This implies that these stretches should be excluded as potential epitopes for our subunit cross-reacting antibodies, thus narrowing down the possible localization of the epitopes and nicely fits with the broad locations we postulated. Furthermore, the fact that the antibodies are weakly positive on a 25-fold increased loading of a Stylonychia fusion peptide starting at position 214, suggests that of the three possible amino-acid domains, the region ct(258-267), ]3(256--265) could, at least partly, constitute the epitope. In that framework, the very weak reactivity of TAP 956 ° and TAP 926G on fusion proteins containing the presumptive epitope could be interpreted as being due either to direct steric hindrance or to an indirect eonformational effect on the epitope caused by the associated large fragment of fl-galactosidase. In summary, the strong or weakly positive reactions over fusion proteins, always corroborated by other data, specially immunocytochemical observations, allows to unambigu-
ously identify antibodies directed to amino-acid sequences, in a broad sense. This class being thus delineated, four other specially interesting types of antibodies which all display several properties different from the ones just described were identified. Antibodies directed against four distinct types of post-translational modifications
These antibodies were totally unreactive over fusion proteins, even if huge amounts are challenged (this is evidenced in fig 4C, AXO 58), in clear contrast with the antibodies classified as 'anti-sequence': they could be directed against either conformational or post-translational epitopes. However, they attracted our attention because of some similarities in the pattern of decoration of Paramecium cells they yielded as compared to those of reference antibodies directed against post-translational modifications. They can be further subdivided into two classes: the first is directed against an epitope the nature of which is identified, acetylation, and the second is directed to still unidentified epitopes. Antibodies to acetylated oc-tubulin. We have very clearly shown that three antibodies TEU 318 °, TEU 348 ° and TAP 931E behave exactly as 6-11B-1 antibody whose epitope is generated by acetylation of lysine 40 on t~-tubulin [44, 45]: they are totally negative on fusion proteins (fig 4B), decorate stable microtubules both in Paramecium and in metazoan cells, fail to label the microtubules of PtK2 cells, and weakly recognize Paramecium cytoplasmic tubulin and that of pig brain. All three, as 6-11B-1, became strongly reactive over chemically acetylated pig brain tubulin. In the conditions used, the main acetylated amino-acids are reported to be lysine and tyrosine residues but the latter were further deacetylated. Through peptide map immunoblotting, the epitope(s) recognized by the three antibodies was (were) located in the 1-100 region of the N-terminal domain of Paramecium ottubulin (no labeling of fl-tubulin peptides was ever observed). This region contains only three lysine residues, at positions 40, 60 and 96. Through direct sequencing, LeDizet and Piperno [44] have established that acetylation occurred on lysine 40 in Chlamydomonas tx-tubulin; an identical localization was found by Edd6 et al [23] on pig brain tubulin. Moreover, given the identity of Paramecium, Chlamydomonas and mammalian tx-tubulin sequences surrounding lysine 40, it is very tempting to conclude that our three antibodies are directed to an acetylation of lysine 40 occurring in Paramecium tubulin. However, we cannot formally exclude that lysine 60 or 96 could be involved. Antibodies to post-translational modifications other than acetylation In contrast to all the anti-sequence and anti-acetylated tubulin antibodies which all clearly decorated only the tip of Paramecium axonemes under our fixation conditions, the three antibodies, TAP 952 °, AXO 58, and AXO 49 ° yield a differential decoration of axonemes (fig 8). The proximal decoration of axonemes by AXO 58 is reminiscent of that provided by the monoclonal antibody GT 335 [10], directed against tubulin polyglutamylation [75]. The strong decoration of axonemes by AXO 49 ° is very similar to that of a previously characterized polyclonal serum raised against Paramecium axonemal tubulin, most probably directed
Tubulin diversity in Paramecium against a post-translational modification [2], while the labeling pattern of axonemes by TAP 952 ° along their whole length is intermediate between the two other ones. In addition, the three antibodies recognize only a specific subset of the other microtubular networks: TAP 952 ° has a very broad reactivity, AXO 58 decorates a more restricted subpopulation of microtubules and AXO 49 ° the narrowest and the most stable one. Finally, and importantly, a lag time is observed between assembly of new microtubular structures after division and their acquisition of reactivity to the AXO 49 ° antibody, as was observed with the polyclonal anti-Paramecium axonemal tubulin serum [2], providing a decisive argument in favour of the idea that it is directed to a post-translationally added epitope. Thus, in order to account for the differential labeling patterns of axonemes, it may be hypothesised that the modification recognized by AXO 49 ° leads to an apparent inacessibility of DM1A, DM1B, o~S, flS and Ac antibodies to their epitopes along the axoneme of Paramecium (except at the tip), ie precisely where the reactivity of AXO 49 ° is found. We have at present localized the presumed post-translational epitopes of three antibodies in a large C-terminal domain of both ¢x- and ~tubulins. Interestingly, it is in this region, at the C-terminal end, that all the known post-translational modifications of tubulin have been discovered (except for acetylation). Such is the case £or: i) detyrosination of metazoan ¢x-tubulin, but no C-terminal tyrosine is encoded by the known sequences of ciliates; ii) phosphorylation of ~tubulin; and iii) polyglutamylation, found in mammalian c~- and ~tubulins respectively at positions 445 and 435 (or 438). Only this modification has been shown to occur on both ¢x- and ~-tubulins, at their C-terminal ends. Some modifications revealed by our antibodies could be of the same nature as these. For example, AXO 58 displays many of the characteristics of the anti-glutamylation antibody GT 335. It is therefore tempting to hypothesize that AXO 58 is directed against tubulin glutamylation, the differences in reactivity pattern with respect to GT 335 being due to the fact that the two antibodies react preferentially with different forms of glutamylation. The same comment can be made for TAP 952 °. In contrast, for AXO 49 ° , the specificity corresponds very closely to that of the anti-Paramecium axonemal tubulin polyclonal serum and all the evidence, partly provided here and detailed in a forthcoming paper (Levilliers et al, in preparation), points to a new type of post-translational modification specially occurring on axonemes and also on a few other very stable microtubules and displaying a broad phylogenetic distribution. Two comments can be made on the differential expression of antigenic sites of tubulin. First, the anti-sequence antibodies we have recovered are directed to epitopes located either in a 30-kDa N-terminal/central ¢x peptide or in a 21-kDa C-terminal fl peptide, which precisely correspond to the most stable domains of tubulin, as reported by Mandelkow et al [49]; these would be long-lived within the immunized animal relative to the other ones, and hence more prone to induce an immune response. Second, the antigenic determinants corresponding to post-translational m o d i f i c a t i o n s and t h o s e c o r r e s p o n d i n g to p r i m a r y sequences seem to be mutually exclusive. Indeed, as conterns the C-terminal domain of axonemal tubulin, which is exposed and probably immunogenic (this being confirmed by the existence of the DM1A and DM1B antibodies and other anti-sequence ones (for review see [4]), we have obtained only one anti-sequence fl and no anti-sequence but three anti (¢x + fl)-tubulin antibodies p r e s u m a b l y
117
directed against distinct post-translational modifications. If these modifications are bulky, they may be immunodominant and could mask the potential sequence determinants, all the more that axonemal tubulin may be highly modified. In conclusion, we have produced a number of anti-tubulln antibodies which are directed either against primary sequences or against post-translational epitopes and we have provided a number of congruent circumstantial evidence indicating that several of the latter antibodies belong to previously undescdbed specificities. While the panel is not exhaustive, it is already sufficiently large and the biochemical characterization of the epitopes is sufficiently advanced for these antibodies to be used as tools of defined specificity both for detailed immunocytochemical studies and for further biochemical characterization of the epitopes. Acknowledgments This work was supported by grants from the CNRS, the 'Universit~ Paris-Sud', the 'Action Inttgrte Franco-Espagnole n° 91047', and the 'Direccion General de Investigacion cientifica y t~cnica (DGICYT), project PB 91/0621'. This work could not have been carried out without the generous gifts of samples and antibodies provided by the many colleagues listed in Materials and methods, to whom we are grateful. We thank Herv6 Le Guyader for this contribution at the start of work, Herr6 Philippe for help with computing and for sharing aligned sequences, and Michel Laurent for his assistance in confocal microscopy and image processing. We would also like to thank No,lie Narradon for all the photographic work, and CEcile Couanon for her extensive help in editing the manuscript. References
1 Adoutte A, Claisse M, Maunoury R, Beisson J (1985) Tubulin evolution: Ciliate-specific epitopes are conserved in the ciliary tubulin of metazoa. J Mol Evo122, 220-229 2 Adoutte A, Delgado P, Fleury A, Levilliers N, Lain6 MC, Marry MC, Boisvieux-UMch E, Sandoz D (1991) Microtubule diversity in ciliated cells: evidence for its generation by post-translational modification in the axonemes of Paramecium and quail oviduct cells. Biol Cell 71,227-245 3 Alexander JE, Hunt DF, Lee MK, Shabanowitz J, Michel H, Berlin SC, Macdonald TL, Sundberg RJ, Rebhun LI, Frankfurter A (1991) Characterization of posttranslational modifications in neuron-specific class HI ~-tubulin by mass spectrometry. Proc Natl Acad Sci USA 88, 4685-4689 4 Andreu JM, de Pereda JM (1993) Site-directed antibodies to tubulin. Cell Motil Cytoskeleton 26, 1-6 5 Axevalo MA, Nieto JM, Andreu D, Andreu JM (1990) Tubulin assembly probed with antibodies to synthetic peptides. J Mol Bio1214, 105-120 6 Barahona I, Soares H, Cyrne L, Penque D, Denoulet P, Rodrigues-Pousada C (1988) Sequence of one ~- and two ~-tubulin genes of Tetrahymena pyriformis. Structural and functional relationships with other tubulin genes. J Mol Biol 202, 365-382 7 Barra HS, Arce CA, Rodriguez JA, Caputto R (1974) Some common properties of the protein that incorporate tyrosine as a single unit and the mierotubule proteins. Biochem Biophys Res Commun 60, 1384-1390 8 Best D, Wart PJ, Gull K (1981) Influence of the composition of commercial sodium dodecyl sulfate preparations on the separation of ~- and ~tubulin during polyacrylamide gel electrophoresis. Anal Biochem 114, 281-284 9 Blose SH, Melzer DI, Feramisco JR (1984) 10-rim filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Bio198, 847-858
118
AM Callen et al
10 Br6 MH, de Ntchaud B, Wolff A, Fleury A (1994) Glutamylated tubulin probed in eiliates with the monoclonal antibody GT 335. Cell Motil Cytoskeleton 27, 337-349 11 Breitling F, Little M (1986) Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL 1/34, DMIA and DMIB. J M o l Biol 189, 367-370 12 Cleveland DW (1987) The multitubulin hypothesis revisited: What have we learned? J Cell Biol 104, 381-383 13 Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel eleetrophoresis. J Biol Chem 252, 1102-1106 14 Cohen J, Adoutte A, Grandchamp S, Houdebine LM, Beisson J (1982) Immunocytochemical study of microtubular structures throughout the cell cycle of Paramecium. Biol Cell 44, 35-44 15 Cohen J, Beisson J (1988) The cytoskeleton. In: 'Paramecium' (Gtrtz HD, ed) Springer-Verlag, Berlin - Heidelberg, 363-392 16 De Jong WW, Zweers A, Cohen LH (1978) Influence of single amino acid substitutions on electrophoretic mobility of sodium dodec.yl sulfate-protein complexes. B i o c h e m Biophys Res Commun 82, 532-539 17 De la Vina S, Andreu D, Medrano FJ, Nieto JM, Andreu JM (1988) T~bulin structure probed with antibodies to synthetic peptides. Mapping of three major types of limited proteolysis fragments. Biochemistry 27, 5352-5365 18 Draber P, Draberova E, Linhartova I, Viklicky V (1989) Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domainspecific monoclonal antibodies. J Cell Sci 92, 519-528 19 Drapeau GR (1976) Protease from Staphyloccus aureus. Methods Enzymo145, 469--475 20 Dupuis P (1992) ~Structure, Organisation et Expression des GEnes de Tubuline chez la Paramtcie~. Doctoral Thesis, Universit6 Paris XI, Orsay 21 Dupuis P (1992) The beta-tubulin genes of Paramecium are interrupted by two 27 bp introns. EMBO J I1, 3713-3719 22 Edd6 B, Jeautet C, Gros F (1981) One ]~-tubulin subunit accumulates during neurite outgrowth in mouse neuroblastoma ceils. Biochem Biophys Res Commun 103, 1035-1043 23 Edd6 B, Rossier J, Le Caer JP, Berwald-Netter Y, Koulakoff A, Gros F, Denoulet P (1991) A combination of posttranslational modifications is responsible for the production of neuronal a-tubulin heterogeneity. J Cell Biochem 46, 134-142 24 Edd6 B, Rossier J, Le Caer JP, Desbruy~res E, Gros F, Denoulet P (1990) Posttranslational glutamylation of atubulin. Science 247, 83-85 25 Eipper BA (1974) Properties of rat brain tubulin. J Biol Chem 249, 1407-1416 26 Fleury A (1991) Dynamics of the cytoskeleton during morphogenesis in the Ciliate Euplotes. I. Basal bodies related microtubular system. Eur J Protisto127, 99-114 27 Fling SP, Gregerson DS (1986) Peptide and protein molecular weight determination by electrophoresis using a high-molarity Tris buffer system without urea. Anal Biochem 155, 83-88 28 Forgue ST, Dalai JL (1979) Rat brain tubulin: tubulin heterogeneity and phosphorylation. J Neurochem 32, 1015-1025 29 Gaertig J, Thatcher TH, McGrath KE, Callahan RC, Gorovsky MA (1993) Perspectives on tubulin isotype function and evolution based on the observation that Tetrahymena thermophila microtubules contain a single ¢x-and ~tubulin. Cell Motil Cytoskeleton 25, 243-253 30 Gallo JM, Anderton BH (1983) A subpopulation of trypanosome mierotubules recognized by a monoclonal antibody to tubuUn. EMBO J 2, 479--483 31 Gard DL, Kirsehner MW (1985) A polymer-dependent increase in phosphorylation of ~tubulin accompanies differentiation of a mouse neuroblastoma cell line. J Cell Biol 100, 764-774 32 Geuens G, Hill AM, Levillier~ N, Adoutte A, DeBrabander M (1989) Mierotubule dynamics investigated by microinjoction of Paramecium axonemal tubulin: Lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase. J Cell Biol 108, 939-953
33 Greer K, Rosenbaum JL (1989) Post-translational modifications of tubulin. In: Cell movement. Vol 2: Kinesin, Dynein and Microtubule Dynamics (Warner FD, Macintosh JR, eds) Alan R Liss, New York, 47--66 34 Grimm M, Breitling F, Little M (1987) Location of the epitope for the cz-tubulin monoclonal antibody TU-01. Biochim Biophys Acta 91, 4 83-88 35 Gull K, Hussey PJ, Sasse R, Schneider A, Seebeck T, Sherwin T (1986) Tubulin isotypes: Generation of diversity in cells and microtubular organelles. J Cell Sci (Suppl) 5, 243-255 36 Helftenbein E, MUller E (1988) Both a-tubulin genes are transcriptionally active in Stylonychia lemnae. Curr Genet 13,425--432 37 Hoyle HD, Raft EC (1990) Two Drosophila beta tubulin isoforms are not functionally equivalent. J Cell Biol 111, 1009-1026 38 Huang J, Matthews HR (1990) Application of sodium dodecyl sulfate-gel electrophoresis to low molecular weight polypeptides. Anal Biochem 188, 114--117 39 Jeanmaire-Wolf R, Cltrot JC, Nahon P, Iftode F, Fleury A, Adoutte A (1993) Isolation and characterization of monoclohal antibodies to cytoskeletal and membrane proteins of the Paramecium cortex. Eur J Protisto129, 311-333 40 Joshi HC, Cleveland DW (1990) Diversity among tubulin subunits: toward what functional end? Cell Motil Cytoskeleton 16, 159-163 41 Kopczak SD, Haas NA, Hussey PJ, Silflow CD, Snustad DP (1992) The small genome of Arabidopsis contains at least six expressed a-tubulin genes. Plant Cell 4, 539-547 42 Kyhse-Andersen J (1984) Eloctroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitroceUulose. J Biochem Biophys Methods 10, 203-209 43 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680--685 44 LeDizet M, Piperno G (1987) Identification of an acetylation site of Chlamydomonas /x-tubulin. Proc Natl Acad Sci USA 84, 5720-5724 45 LeDizet M, Piperno G (1991) Detection of acetylated a-tubulin by specific antibodies. Methods Enzymol 196, 264-274 46 L'Hemault SW, Rosenbaum JL (1983) Chlamydomonas ottubulin is posttranslationally modified in the flagella during flagellar assembly. J Cell Bio197, 258--263 47 L'Hemault SW, Rosenbaum JL (1985) Chlamydomonas ottubulin is posttranslationally modified by acetylation on the e-amino group of a lysine. Biochemistry 24, 473--478 48 Luduena R.F, Zimmermann HP, Little M (1988) Identification of the phosphorylated ~tubulin isotype in differentiated neuroblastoma cells. FEBS Lett 230, 142-146 49 Mandelkow EM, Herrmann M, Rtihl U (1985) Tubulin domains probed by limited proteolysis and subunit-specific antibodies. J Mol Biol 185, 311-327 50 Merril CR, Goldman D, Sedman SA, Ebert MH (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437-1438 51 Merril CR, Goldman D, Van Keuren ML (1983) Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymo196, 230-239 52 Paturle-Lafanech~re L, Edd6 B, Denoulet P, Van Dorsselaer A, Mazarguil H, Le Caer JP, Wehland J, Job D (1991) Characterization of a major brain tubulin variant which cannot be tyrosinated. Biochemistry 30, 10523-10528 53 Pipet'no G, Fuller MT (1985) Mouoelonal antibodies specific for an acetylated form of ¢x-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol 101, 2085-2094 54 Piperno G, LeDizet M, Chang X (1987) Microtubules containing acetylated ¢x-tubulin in mammalian cells in culture. J Cell Biol 104, 289-302 55 Raft EC (1984) Genetics of mierotubule systems. J Cell Biol 99, 1-10
Tubulin diversity in Paramecium 56 Raybin D, Flavin M (1975) An enzyme tyrosilating alphatubulin and its role in microtubule assembly. Biochem Biophys Res Commun 65, 1088-1095 57 Redeker V, Melki R, Prom6 D, Le Caer JP, Rossier J (1992) Structure of tubulin C-terminal domain obtained by subtilisin treatment. The major t~ and fl tubulin isotypes from pig brain are glutamylated. FEBS Lett 313, 185-192 58 Riordan JF, Vallee BL (1967) Acetylation. Methods Enzymol I 1,565-570 59 Riordan JF, Vallee BL (1967) O-Acetyltyrosine. Methods Enzymol 11,570-576 60 Rtidiger M, Plessman U, Kl6ppel KD, Wehland J, Weber K (1992) Class 1I tubulin, the major brain fl tubulin isotype is polyglutamylated on glutamic acid residue 435. FEBS Lett 308, 101-105 61 Sasse R, Gull K (1988) Tubulin post-translational modifications and the construction of microtubular organelles in Trypanosoma brucei. J Cell Sci 90, 577-589 62 Schliwa M, van Blerkom J (1981) Structural interaction of cytoskeletal components. J Cell Bio190, 222-235 63 Serrano L, Wandosell F, Avila J (1986) Location of the regions recognized by five commercial antibodies on the tubulin molecule. Anal Biochem 159, 253-259 64 Silflow CD, Oppenheimer DG, Kopczak SD, Ploense SE, Ludwig SR, Haas N, Snustad DP (1987) Structure and differential expression during development. Dev Genet 8, 435--460 65 Steinbrtick G (1990) Recent advances in the study of ciliate genes. Eur J Protisto126, 2-14 66 Sullivan KF (1988) Structure and utilization of tubulin isotypes. Annu Rev Cell Biol 4, 687-716
119
67 Suprenant KA, Hays E, l..eCluyse E, Dentler WL (1985) Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila. Proc Natl Acad Sci USA 82, 6908---6912 68 Swank RT, Munkres KD (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem 39, 462--477 69 Thompson WC (1982) The cyclic tyrosination/detyrosination of alpha tubulin. Methods Cell Bio124, 235-255 70 Torres A, Delgado P (1989) Effects of cold and nocodazole treatments on the microtubular systems of Paramecium in interphase. J Protozoo136, 113-119 71 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76, 4350--4354 72 Tung JS, Knight CA (1972) Relative importance of some factors affecting the electrophoretic migration of proteins in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 48, 153-163 73 Viklicky V, Draber P, Hasek J, Bartek J (1982) Production and characterization of a monoclonal antitubulin antibody. Cell Biol Int Rep 6, 725-731 74 Wolff A, de N6chaud B, Chillet D, Gros F (1988) The aminoterminal domain of alpha-tubulin probed by monoclonal antibodies. Recognization of a rarely exposed epitope by the monoclonal antibody 111 B52 C2. Biol Cell 63, 319-326 75 Wolff A, de N6chaud B, Chillet D, Mazarguil H, Desbruy~res E, Audebert S, Edd6 B, Gros F, Denoulet P (1992) Distribution of glutamylated tx and fl-tubulin in mouse tissues using a specific monoclonal antibody, GT 335. Eur J Cell Bio159, 425--432
95
© Elsevier, Pads
Original article
Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium Anne-Marie Callen a, Andr6 Adoutte a, Jose Manuel Andrew b, A n n e Baroin-Tourancheau a, Marie-H61~ne Br6 a, Purificacion Calvo Ruiz e, Jean-Claude C16rot a, Pilar Delgado a, A n n e Fleury a, Rachel Jeanmaire-Wolf a, Vladimir Viklicky d, Eduardo Villalobo c, Nicolette Levilliers a aLaboratoire de Biologie Cellulaire 4, URA l134-CNRS, Bdtiment 444, Universitd de Paris-Sud, 91405 Orsay Cedex, France; b Centro de Investigaciones Biologicas, c/o Velazques 144, 28006 Madrid; bDepartarnento de Microbiologia, Facultad de Biologia, Universidad de Sevilla, Apdo 1095, 41080 Sevilla, Spain; dlnstitute of Molecular Genetics, Czechoslovak Academy of Sciences, Videnska 1083, 14220 Prague 4, Czech Republic (Received 28 June 1994; accepted 18 July 1994)
Summary - Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and I0 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the t~ N-terminal domain, one in the fl C-terminal one, and two in tx and fl central stretches). The remaining ones decorated only a specific subset of rnicrotubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of wtubulin whereas the epitopes of three other ones (TAP 952 °, AXO 58 and AXO 49 °) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both ix- and fl-tubulins. The AXO 49 ° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences. ciliate tubulin / axonemal tubulin / tubulin epitopes / tubulin post-translational modifications / immunofluorescence
Introduction Two recurrent questions in microtubule research have been those of the origin of tx- and.fl-tubulin diversity and its functional significance. Concerning the first question, much progress has been made during the last few years with the realization that tubulin diversity can be attributed both to primary genetic redundancy of tubulin coding genes and to a variety of post-translational modifications. The inventory of both gene diversity and the biochemical bases of post-translational modifications has advanced in a variety of organisms, especially metazoans and metaphytes, but also in several protists and fungi (see [12, 33, 35, 41, 55, 64, 66] for reviews). For metazoans, the picture usually emerging has been that of a fairly high diversity of genes coding for a variety of isotypes often expressed in a tissue-specific pattern coupled to at least five well identified post-translational modifications: acetylation of wtubulin on lysine 40 [23], phosphorylation of a fl-tubulin isotype near the C-terminal end [3, 22, 25, 28, 31, 48], polyglutamylation of both ct- and fl-tubulins near the C-terminal extremity [3, 24, 57, 60], detyrosination of the C-terminal end of t~-tubulin [7, 56, 69]
and removal of the glutamyl tyrosine dipeptide at the C-terminal end of tx-tubulin [52]. In protists, especially in ciliated or flagellated species, one m a j o r difference has been observed in terms of genetic redundancy with respect to multiceUular organisms: a- and fl-tubulins are each encoded by a very small number of extremely similar (if not identical) genes (at the amino-acid level). A striking example is that of the ciliate Tetrahymena which builds its large variety of microtubular networks from only one a and two almost (Tpyriformis [6]) or completely (T thermophila [29]) identical fl gene products. In contrast, post-translational modifications in protists appear to be very similar to those identified in multicellular organisms: in particular acetylation has been discovered in one protist [44, 46, 47] and then extended to a variety of protists and other organisms (reviewed in [45]). As for glutamylation, it has just been discovered in ciliates [10]. In addition, one notable, still biochemically uncharacterized post-translational modification has been found to occur in Paramecium axonemes and later in those of a very large array of organisms extending from protists to mammals [1, 2]. Except for trypanosomes [61], evidence for detyrosination is lacking at the moment in other protists.
96
AM Callcn et al
Concerning the second question, unequivocal demonstrations of the function of microtubule primary and secondary diversity are scarce to date ([37], for a review see [40]). Ciliates and specially Paramecium are well adapted for analysis of the processes through which diversification of initially identical microtubules takes place within the same cytoplasm. Indeed, at least 13 distinct microtubular arrays have been described in Paramecium [15], and although the number of tx- and fl-tubulin genes is higher in Paramecium than in Tetrahymena (respectively four and three), all the sequence work carded out so far shows that the resulting proteins are e x t r e m e l y similar [20, 21]. Thus in this system, most if not all, differences in the properties of the diverse microtubular arrays can be attributed to post-translational modifications and/or interactions with accessory proteins. So far, the immunocytochemical analysis of microtubule diversity in Paramecium [2, 10, 15, 70] has mostly relied on well characterized antibodies raised in other systems such as the 'universal' anti c~- and anti-fl-tubulin antibodies D M 1 A and DM1B [9] reacting respectively on tx- and fl-tubulin primary sequences [11], or antibodies directed against well identified post-translational modifications such as 6-11B-1 which reacts with acetylated tx-tubulin [53], or GT 335 which recognizes glutamylated tubulin [75]. In addition, a polyclonal serum raised against Paramecium axonemal tubulin [14] was used and it was recently suggested that its major epitope corresponds to a post-translational modification different from others [2]. In the present work, we have approached the question of microtubule diversity in Paramecium by directly isolating libraries of monoclonals to ciliate tubulins, aiming at recovering as large a spectrum as possible. We describe the various fusions carried out and the characterization of eight monoclonal antibodies and 10 hybridoma supernatants in terms of specificity over a- and fl-tubulins, interaction with non-ciliate tubulins, location of the epitope as deduced from reactivity on peptide maps and from sequence comparisons between ciliate and vertebrate tx- and fl- tubulins, and, finally, identification of the epitope as being located in the primary sequence of tubulin or being due to a post-translational modification. This last distinction was made through the presence or absence of recognition over fusion proteins generated by genetic engineering in E coli: it was based on the idea that most (if not all) of the modifications known to occur, on tubulin do not take place in E coli and even if they did, it would be highly unlikely that the E coli modification enzymes would recognize a typically eukaryotic protein such as tubulin. We also document the irnmunocytochemicai properties of the various antibodies. It will be seen that a very broad collection of antibodies has been obtained, apparently revealing an extensive microtubular diversity existing in Paramecium; these antibodies will provide powerful tools for analysis of both the nature and genesis of post-translational modifications and of the patterning of microtubules during morphogenesis.
Materials and methods Strains and cell cultures Paramecium tetraurelia cells (strain d4-2) were grown at 27°C in phosphate buffered Wheat Grass Powder infusion, supplemented with 0.4/2g/ml fl-sitosterol and baeterized with Klebsiella pneumoniae. Euplotes aediculatus and Stylonychia lemnae (a generous gift from Drs E Helftenbein and M Schlegel)
were fed with Tetrahymena cells grown axenically according to Fleury [26]. 3T3 cells were cultured in RPMI medium supplemented with 2 mM glutamine, 100 IU/ml penicillin and 100 /ag/ml streptomycin, then grown on glass coverslips for immunofluorescence. PtK2 epithelial cells were grown as described by Geuens et al [32]. Antigens Cytoskeletal tubulin of Euplotes Euplotes cells were treated in PHEM buffer, at pH 6.9 [62], supplemented with 0.5 Triton X-100, leupeptin (4/zM') and PMSF (0.3 mM) for 5 rain. Extracted cells were boiled for 3 min in electrophoresis sample buffer and loaded onto 7.5-15% acrylamide gradient, 12-cm long, 3-mm thick, preparative SDS-polyacrylamide gels [43]. After electrophoresis, tubulin was visualized after a 0.25 M KCI treatment of the gels and the acrylamide bands were excised; tubulin was electroeluted in a 0.025 M TrisHCI, 0.192 M glycine buffer at pH 8.3 containing 0.025% SDS, using a Biotrap apparatus from Schleicher and Schuell (Ecquevilly, France). It was kept frozen at -200C. Paramecium axonemes Prepared according to Geuens et al [32] and kept frozen at -80°C. Axonemal tubulin of Paramecium Axonemes were submitted to preparative SDS-PAGE. Tubulin was electroeluted and stored as described for Euplotes. Production of monoclonal antibodies
BALB/c mice immunizations, fusions, hybridoma screening and sub-cloning were carded out as described by Jeanmaire-Wolf et al [39] with minor modifications as detailed in table I. ELISA development was made using either a secondary sheep antimouse IgG antibody coupled to peroxidase (Diagnostics Pasteur, France) and, as substrate, 0.1% ortho-phenylene diamine with 0.002% 1-I202or a rabbit anti-mouse IgG labeled with fl-galaetosidase (Biosys, Compi~gne, France) and ortho-nitrophenyl-fl-ogalactoside (Sigma, St Louis, MO, USA) as chromogen. Protein preparations Construction offusion proteins Construction of fl-galactosidase a-tubulin fusion proteins. A recombinant plasmid (pBR 322) containing the c(2-tubulin gent from Stylonychia lemnae [36] was kindly provided by F Caron (Ecole Normale Sup6rieure, Paris, France). Different portions of the a-tubulin gene were cloned in-frame behind DNA encoding fl-galactosidase in the pEX expression vectors (Boehringer, Mannheim, Germany). The coding sequence of this gene contains the codon UAA (position 177) which codes for glutamine in many ciliates [65]. Expression of the inserts containing this codon gives thus truncated fusion proteins. The different hybrid fl-galactosidase constructions were designed so that the STOP codon was never located upstream of the insert to be expressed. The expressed inserts correspond to the following domains of the a-tubulin protein: one segment located towards the N-terminal side of Stylonychia ~-tubulin, N, (amino-acids 22-176) and another one comprising the C-terminal side, C (21A. 445) (fig 3). To express the internal domain (177-214), a distinct construction experiment has been performed. Expression of the hybrid proteins. Expression of the hybrid fl-galactosidase is under the control of a ,t.-Pr promoter. Transformations were performed using pop 2136, a host strain carrying the temperature sensitive lambda CI repressor allele. Recombinant plasmids were amplified at 30°C and their transient expression was induced by shifting to 42"C. After 1 or 2 h at 42*C, 1 ml of the culture was removed. The pelleted bacteriai cells were resuspended in sample buffer, boiled for 5 min and submitted to SDS-PAGE for Western blot analysis.
Tubulin diversity in Paramecium
97
Table I. Overview of immunizations and fusion results. The names which are indicated are those of antibodies which have been chosen
for this study; their characteristics are recapitulated in table II.
Name offusion Antigen
Interval between boosts
TEU
Electroeluted Euplotes Electroeluted Paramecium cytoskeletal tubulin axonemal tubulin (tubulin from (tubulin from Euplotes = TEU) axonemes of Paramecium = TAP) 3 weeks 10 weeks 3 weeks 20 weeks + 4 days: 1 fusion + 3 days: 1 fusion
Number of hybridoma containing wells tested Number (and percent) of positive ELISA tests
Tests carried out and criteria for storing Major characteristics of hybridoma supernatant tested; decoration of: - all microtubular networks - stable microtubules - cilia along all their length and all microtubular networks - cilia, except at their tip
AXO Paramecium axonemes (= AXO) 1 week 4 weeks + 4 days: 1 fusion
864
480
480
118 (13.6%)
65 (13.5%)
91 (18.9%)
1. ELISA on Euplotes, - ELISA on Paramecium - ELISA on axonemes, Euplotes Paramecium, pig brain tubulin and pig brain tubulin and pig brain tubulin hybridoma retained if strongly or moderately posititve in ELISA on Euplotes and/or Paramecium tubulin 2. Immunofluorescence over whole cells of Paramecium; ~ hybridomas stored if: - clear decoration a - decoration of cilia of various microtubular networks b - various decoration patterns of intracellular networks Number Name Number Name Number Name 19 2 0
TEU 435*, TEU 310 °, TEU 441, TEU 4211G, TEU 4211H, TEU 4312 TEU 318 °, TEU 348 °
0
Expression of a fl-galactosidase-ot-tubulin fusion protein containing the domain (177-214). A copy of the ¢x-tubulin gene from Euplotes aediculatus was amplified using a couple of degenerate primers. The amplification product, 1.2 kb long, was cloned blunt ended in the phagemid pBS (Stratagene, La Jolla, CA, USA) in-frame with the DNA encoding the N-terminal part of the ~-galactosidase. In Euplotes aediculatus the sequence of the gene does not contain any STOP codon since this species appears to use the u n i v e r s a l code [65]. In this case, the expressed insert corresponds to a large fragment covering the almost complete sequence of the protein, E (25--412) (fig 3). Expression by this plasmid is controlled through a promoter that is activated with isopropyl-/~-D-thiogalaetopyranoside (IPTG). In the strain XL1 blue transformed with the recombinant plasmid, induction was induced by addition of 5 mM IPTG. After a 4-h incubation in presence of IPTG, the bacterial cells were treated as described above.
Cell extracts Stylonychia cytoskeletons.
TAP
4 1 2
TAP 9311, TAP 931B, TAP 926, TAP 956 ° TAP 931E TAP 952 °
0
4
AXO 16, AXO 45 °
5 0
AXO 58
3
A X O 49*
diluted to 1 mg/ml in 1 M sodium phosphate at pH 8 supplemented with 1 mM GTP and 1 mM MgCI2; acetic anhydride was added under constant stirring, at room temperature, by 2-/.d aliquots, every 5 rain while maintaining a constant pH (8-8.5) by addition of 1 N NaOH. The final acetic anhydride concentration was 1% and the whole procedure took 30 rain. Therefore, under these conditions, most e-aminogroups of lysine and also tyrosine residues should be acetylated. The latter can be deaeetylated by hydroxylamine at neutral pH. To this purpose, after dialysis of the sample, hydroxylamine in 20 mM sodium phosphate at pH 7 was brought to 0.1 M in the acetylated tubulin sample; this solution was kept under constant stirring during 30 rain and then dialyzed. Acetylated tubulin was frozen in liquid nitrogen.
Reference anti-tubulin antibodies Monoclonals
Stylonychia cells were treated with
DMIA and DM1B [9] were purchased from Amersham (Les Ulis, France); TU-01 [73] was used; 6-11B-1 culture supematant [53] was a gift of Dr G Piperno (The Rockefeller University, New York, USA) whereas 6-11B-I ascitic fluid was purchased from Sigma; 111 B52 C2 [74] and GT 335 [75] were generously provided by Dr A Wolff (Coll~ge de France, Paris).
0.5% Triton X-100 in PHEM buffer supplemented with 4/zM leupeptin for 5 rain. Extracted cells were boiled in electrophoresis sample buffer and kept frozen in aliquots at -80°C. PtK2 cells. Confluent PtK2 cells were rinsed with PBS, then scrapped from Petri dishes, and boiled for 5 min in electrophoresis sample buffer supplemented with 8/AM leupeptin and 2 mM PMSF and kept frozen at -20°C. Paramecium cytoplasmic extract. Prepared according to Br6 et al [I0]. Phosphocellulose purified porcine brain tubulin. Kindly provided by Dr MF Carlier (CNRS, Gif-sur-Yvette, France).
Immunofluorescence microscopy
Chemical acetylation of porcine brain tubulin. Acetylation was carried out according to Riordan and Vallee [58, 59] and Piperno and Fuller [53] with some modifications: tubulin was
For Paramecium, the immunofluorescence protocol described in Jeanmaire-Wolf et al [39] was followed with minor modifications: the permeabilization buffer (PHEM at pH 6.9 with 1% Tri-
Polyclonals Affinity-purified C 98, C 140 and C 113 monospecific IgG antibodies [5] were employed.
98
A_M Callen et al
ton X-100) was supplemented with leupeptin (4/tM) and the ghosts were fixed in freshly dissolved 2% paraformaldehyde, in PHEM buffer for 45 min. After washes, they were incubated in one anti-tubulin antibody and then in the fluorescein-conjugated sheep-anti-mouse IgG antibody (Diagnostics Pasteur, France) diluted 1:200 and rinsed. Cells were mounted using Citifluor (London) and examined on a Bio-Rad Lasersharp MRC 600 confocal microscope equipped with an argon laser. For 3T3 and PtK2 cells, two fixation procedures were used: either with methanol at -20°C for 3 rain or with 1/10 formaldehyde (commercial solution) for 30 min in PBS followed by 50 mM NH4CI in PBS (2 × 5 rain) and 0.1% Triton X-100 in PBS (10 rain). Cells were rinsed in PBS, saturated with 3% BSAPBS buffer, incubated with primary antibodies diluted in 3% BSA-PBS for 90 rain, further incubated with fluorescein-conjugated sheep-anti-mouse IgG (1:50 in 3% BSA-PBS for 30 rain) and processed like Paramecium cells. Gel electrophoresis and immunoblotting SDS-PAGE was conducted according to Laemmli [43]. Briefly, proteins were boiled for 3 rain in Laemmli's sample buffer and loaded on 0.75-ram thick, 10% or 8% polyacrylamide mini-gels (unless otherwise mentioned) containing a lower gel buffer respectively adjusted to pH 8.2 or pH 9.3 for Paramecium axonemes and fusion proteins or porcine brain tubulin. SDS from BDH Laboratory Supplies (Poole, UK) was mostly used [1]. After electrophoresis, gels were either stained by Coomassie brilliant blue R 250 or electro-transferred onto 0.45//m nitrocellulose filters using a semi-dry transfer CarboGlas apparatus (Schleicher and Schuell) and the protocol described by Kyhse-Andersen [42] (with methanol replaced by isopropanol in the transfer buffers). Membranes were stained With Ponceau red, destained, saturated in PBS-3% BSA for 1 h at 37°C, further incubated for 1 h at 37°C (or overnight at room temperature) with primary antibodies diluted in PBS-0.25% BSA-0.1% Tween 20, and extensively washed in the antibody buffer. For IgG detection, filters were incubated for 1 h at room temperature with an alkaline phosphatase-labeled goat anti-mouse IgG at a dilution of 1:7500 (Promega, Madison, WI, USA) or with a peroxidase-labeled sheep anti-mouse IgG secondary antibody at 1:2000 (Diagnostics Pasteur). For IgM detection, two successive incubations in class-specific secondary antibody then in labeled enzyme, were used. They involved biotlnylated goat anti-mouse IgM at 1:200 (Amersham), followed by alkaline phosphatase-conjugated streptavidin at 1:1000 (Immunotech, Marseille, France). Development of alkaline phosphatase was carried out with NBT (nitro blue tetrazolium) and BCIP (5-bromo-4-chloro-3indolyl phosphate) as substrates (provided by Sigma). Development of peroxidase was performed with the ECL system from Amersham. Dot-blotting Antigens (2/11 in Laemmli's sample buffer) were dotted on 0.45 p.m nitrocellulose, the membrane was air-dried and saturated in PBS-3% BSA, then treated with antibodies similarly as after electrophoresis and electro-transfer. Peptide mapping In a first step, ~x- and/3-tubulin bands from 10 to 20/~g Paramecium axonemes were separated then cut out from Coomassie blue-stained 12-cm long, 0.75-ram thick 8% polyacrylamide gels [43] performed: i) either, at pH 8.25, with 0.1% specially pure (98-99%) SDS from BDH; or ii) at pH 9.25, with 0.1% SDS from Sigma (70% pure). The respective running buffers contained the corresponding SDS. In the presence of pure SDS at pH 8.25, Paramecium a-tubulin migrates faster than ~tubulin as reported for Tetrahymena tubulin [67], whereas, at pH 9.25, both subunits comigrate. However, they are resolved with impure SDS at pH 9.25 but their relative migrations are reversed and become similar to those of brain tubhlin subunits. This altered behaviour is probably due to the contaminants of SDS, myristil (C14) and eetyl (C16) sulfates, which have been shown to achieve a good separation of brain a- and fl-tubulins [8].
In a second step, the tubulin bands (kept at - 2 0 ° C ) were loaded in the wells of an 1l-cm long, l-ram thick resolving gel made of a 15-20% acrylamide gradient in the presence of BDH SDS: i) either at pH 8.25, in standard Laemmli conditions (figs 6A, 9, 10 and 12); or ii) at pH 8.8, by doubling the "Iris concentration in the lower gel and running buffers, as described by Fling and Gregerson [27] to improve the resolution of small peptides (fig 6B). The tubulin bands were submitted to mild proteolysis by Staphylococcus aureus V8 protease, type XVII-B (Sigma), in the presence of 0.3% 2-mereaptoethanol, during a 2-h migration in the stacking gel (3 cm long) followed by a 1-h arrest of proteins at the interface of the stacking and resolving gels (slightly modified after Cleveland et al [13]). Rainbow coloured protein molecular mass markers (range 2.35-46 kDa) were from Amersham. After overnight electrophoresis, the gels were silver stained according to Merril et al [50], followed by intensification adapted from Merril et al [51]. Briefly, the silver coloured gels were completely destained by soaking for 5-20 rain in a 0.5% solution of Lumiere reducer (a photochemical reagent containing potassium ferricyanide), and after extensive washes in water, were restained by direct incubation in silver nitrate followed by development with AnalaR formaldehyde (BDH). Milli Q filtered water (Millipore, Saint-Quentin-en-Yvelines, France) was used throughout. Alternatively, the gels were electro-transferred onto 0.1 /tin pore size nitrocellulose (Schleicher and Schuell) according to Towbin et al [71], except for methanol replaced by ethanol in the transfer buffer. After Ponceau red staining, destaining and saturation, the filters were incubated overnight in antibodies (diluted in the same solution as for immunoblotting), followed by a 1-h incubation in biotinylated goat anti-mouse IgG or biotinylated donkey anti-rabbit IgG at a dilution of 1:1600. After a further 1-h incubation in streptavidin-biotinylated horseradish peroxidase complex at 1:1 600, detection was achieved using the ECL system. All incubation steps were performed at room temperature and separated by exhaustive 1-h rinsings as for immunoblotting. Antibodies were provided by Amersham.
R ~ Overview o f immunizations and fusions results (tables I, II) The objectives of the successive fusions differed (table I). Initially, we were interested in recovering the full diversity of possible antibodies to tubulin and thus used cytoskeletal tubulin o f E u p l o t e s as i m m u n o g e n in which a m i x t u r e o f T r i t o n - r e s i s t a n t c y t o p l a s m i c , c o r t i c a l and axonemal tubulins is expected to be present; this corresponds to fusion TEU. In the two later fusions, we were specifically interested in recovering monoclonals yielding similar decorations o f cilia and stable microtubules as those given by a rabbit p o l y c l o n a l serum obtained by injecting P a r a m e c i u m a x o n e m a l tubulin in the form of acrylamid bands [2, 14]. Therefore, either electroeluted P a r a m e c i u m a x o n e m a l tubulin (fusion T A P ) or w h o l e a x o n e m e s w i t h o u t any e l e c t r o p h o r e t i c purification o f tubulin and denaturing step were also injected (fusion
~o). Ciliate tubulin, whatever the form under which it was injected to mice, proved to be immunogenic. Effectively, the sera of all mice tested (22) recognized tubulin after the third injection both on immunoblots and in immunofluorescence tests where it decorated a variety of microtubular networks and/or cilia o f P a r a m e c i u m and Euplotes. The percentage o f E L I S A positive h y b r i d o m a supernatants was respectively 13.6, 13.5 and 18.9%, a fairly high proportion considering that tubulin is an evolutionary conserved protein.
21
22
A
19 20
TAP 952 °
AXO58
A X O 49* GT 335*
6-11B-l* TEU 318 ° TEU 348 ° TAP 931E
T A P 956*
14
15 16 17 18
TAP 926G
DMIB* A X O 45 °
DMIA* TU01* TAP 9311 TAP 931B T E U 435* TEU 310 ° T E U 441 TEU 4211G TEU 4211H TEU 4312 A X O 16
name
13
2 12
3 4 5 6 7 8 9 10 11
1
and
IgG1
IgGl
IgG1 IgG1
IgG1 IgG1 IgG1 IgG1
IgG1
IgGl
IgG1 IgG1
IgG1 IgG 1 IgG1 IgG3 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgM
Type
ix+13
13
ix+13 ix+13
. . fl a n d / o r ixA
e e e e
ix + 13
ix + 13
ix + 13 13 a n d / o r ixA
ix ix ix ix
ix + ]3
ix + 13
fl 13
a a ix a ix ix a a a ix ix
Paramecium tubulin axonemal cytoplasmic
. .
.
-
-
-
+ + + + + + + + + +
.
.
.
-
e
e
-
+
.
.
.
-
-
-
+ + + + + + + + + +
lmmunobloting Fusion proteins E C N
.
.
•
ix
ix ix ix ix
ix+13
ix
fl 13
a ix ix ix ix ix
0
0
0 I
1/10 1/5 1/5 < 1/10
1/i0
1/10
1/i0
1 1 < 1/20 0 0 0 0 < 1/20 1/10
Pig brain tubulin Blot Dot reactivity
+ + + +
-
.
.
Ac Ac Ac Ac
+ +
+ . .
+
.
.
.
.
.
.
-
+
.
.
+ Ac
+ Ac
-
+ +
-
-
+ + -
+
.
.
.
.
+
+
+
-
+
-
-
-
. + +
-
+
+ +
+ + -
+ + + +
+
ix ( 1 5 9 - 2 8 1 ) fl ( 1 8 8 - 3 1 0 ) ix ( 1 5 9 - 2 8 1 ) 13(188-310) ix ( L y s 40)* ix ( 1 - 1 0 0 ) ix ( 1 - 1 0 0 ) ix ( 1 - 1 0 0 )
13 ( 4 1 6 - 4 3 0 ) * 13 (257---442)
ix (426--430)* tx ( 6 5 - 9 7 ) * ix ( 9 8 - 1 3 6 ) ix ( 9 8 - 1 3 6 ) a (98-136) ix ( 9 8 - 1 3 6 ) a (98-136) ix ( 9 8 - 1 3 6 ) ix ( 9 8 - 1 3 6 ) ix ( 9 8 - 1 3 6 )
Epitope location
ix (254 449) 13 (257--442)
13(257 4~2)
ix (Glu 445)* 13 (Glu 435 or 438)* ix (254 449)
Immunofluorescence lnterphasic metazoan cells 3T3 PtK2 Form-T Methanol Form-T Methanol
i n d i c a t e d in bold a n d d e s i g n a t e d b y °. * R e f e r e n c e antibodies, the e p i t o p e s o f w h i c h are well k n o w n o n v e r t e b r a t e t u b u l i n s ; literature r e f e r e n c e s : D M 1 A * , D M 1 B * [11]; T U - 0 1 * [44]; G T 3 3 5 * [3, 24, 57, 60, 75]. B l o t t i n g o n Paramecium t u b u l i n a n d f u s i o n proteins: e, v e r y w e a k s t a i n i n g (see [10]). F u s i o n p r o t e i n s : E, C, N; s e e s c h e m a t i c d i a g r a m (fig 3). l e g e n d to f i g u r e 2. I n t e r p h a s i c m e t a z o a n cells: F o r m - T , f o r m o l - T r i t o n o r m e t h a n o l i n d i c a t e t h e f i x a t i o n p r o c e d u r e ; +, s t r o n g l a b e l i n g ; - , w e a k , a s p e c i f i e l a b e l i n g or n o decoration; t u b u l i n a n t i b o d y typical labeling. E p i t o p e location: inferred f r o m reactivity o n p e p t i d e m a p s a n d f u s i o n p r o t e i n s (see fig 3).
B
Anti P T M C
Antiacetylated ix-tubulin: A c
Anti ix+13 tubulin sequence
Anti fltubulin s e q u e n c e : flS
Anti atubulin s e q u e n c e : ixS
Classification
n e d a n t i b o d i e s are [18, 34]; 6 - 1 1 B - 1 " D o t reactivity; s e e Ae, anti-aeetylated
Table I I . S u m m a r y o f t h e c h a r a c t e r i s t i c s o f t h e a n t i b o d i e s . D e n o m i n a t i o n s : ixS, flS, A c , P T M ( p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n ) . N u m b e r s 1, 2, etc, c o r r e s p o n d to t h o s e e m p l o y e d in f i g u r e 2. Sub-clo-
100
AM Callen et al •
x
i
•
p
°
.
.
°"
"x
-'..
:" "" ,."
• S" .. -D :., ,-I,
.~
:" ': '~,•" -
:.
',
'4'.:]:
-
2
,
% !I
.
.
.
.
Of the 18 antibodies described in this paper, eight were successfully sub-cloned; the others remained at the stage of hybridoma supernatant because antibody production was lost upon thawing or sub-cloning. They are mentioned nonetheless because they not only yield interesting results, but they also appear to be truly monoclonal by several criteria. All the antibodies belong to the IgG class except A X O 16, an IgM. All the IgGs belong to the IgG1 subclass except TAP'931B which belongs to the IgG3 one (table ID. In addition to immunocytochemical analysis on Paramecium and metazoan cells, the hybridoma supernatants were submitted to detailed characterization through immunoblotting and immunocytochemical experiments which allowed to group the antibodies into distinct categories presented below. The characteristics o f the eight monoclonal antibodies "(designated by an ° throughout the text) and the 10 hybridoma supematants are recapitulated in table II.
Fig 1. Two projections of a Paramecium decorated with the TEU 435 ° antibody (1:5) and observed by confocal microscopy, a. Ventral side. b. Dorsal side. The antibody decorates all the microtubules, ie the postoral fiber (Po) associated with the oral area (O), the microtubules associated with the contractive vacuole systems (large arrows), as well as the intracytoplasmic network which appears densely ramified (arrows). In addition, the tips of the cilia are strongly stained (arrowheads), while the rest of the axonemal structure is only faintly decorated. Scale bars, 25/.tm.
Antibodies reacting conspicuously with fusion proteins Anti-~-tubulin sequence (ctS) antibodies Nine of the monoclonal antibodies and hybridoma supematants proved to be positive on fusion proteins indicating that they are directed against an tx-tubulin sequence determinant; they were characterized in detail (table II): T A P 9311, TAP 931B, T E U 435 °, TEU 310 °, T E U 441, T E U 4211G, TEU 4211H, T E U 4312, and A X O 16. In immunofluorescence tests carried out on whole ceils o f P a r a m e c i u m , all the antibodies o f this class yield the same decoration pattern as the 'universal' anti-sequence a n t i b o d i e s D M I A and D M 1 B : t h e y d e c o r a t e all the m i c r o t u b u l a r n e t w o r k s e x c e p t for the cilia w h i c h are stained only at their tip under the fixation conditions used (fig 1 a, b). All these antibodies recognize only tx-tubulin on immu-
Fig 2. A, B. Immunoblotting analysis of Paramecium axonemes (A) and pig brain tubulin (B) with monoclonal antibodies and hybridoma supematants. The relative mobilities of cz- and fl-tubulias of both species visible on Coomassie blue stained gels (lanes CB in A and B) are inversed under the different gel conditions used (lower gel at pH 8.2 or 9.3). In A, 1.8/~g axonemes were loaded for Coomassie blue staining, and 0.2/.tg/well for immunoblotting. In B, 0.1/.tg of porcine brain tubulin were loaded in all wells, which is equivalent to the amount of Paramecium tubulin, assuming that axonemes contain 50% tubulin. Filters were incubated with antitubulin antibodies at dilutions listed below• Reference antibodies are quoted with * and monoclonals with o. Lanes 1-14: anti-sequence antibodies; 1, DM1A*: 1:1000; 2, DM1B*: 1:800; 3, TAP 9311: 1:100; 4, TAP 931B: 1:10; 5, TEU 435°: 1:100; 6, TEU 310°: 1:100; 7, TEU 441: 1:200; 8, TEU 4211G: 1:20; 9, TEU 4211H: 1:10; 10, TEU 4312: 1:10; 11, AXO 16: 1:104; 12, AXO 45°: 1:50; 13, TAP 926(3: 1:20; 14, TAP 956°: 1:100; lanes 15-22: other antibodies; 15, 6-11B-1": 1:100; 16, TEU 318°: 1:500; 17, TEU 348°: 1:250; 18, TAP 931E: 1:50; 19, AXO 49°: 1:3; 20, GT 335*: 1:250; 21, AXO 58: 1:2; 22, TAP 952°: 1:100. Revelation was performed with alkaline-phosphate secondary antibodies and NBT/BCIP. C. Dot-blotting analysis of antibody re.activities towards pig brain tubuiin in comparison with Paramecium axonemes. Line AXO Ix: 0.2/.tg of Paramecium axonemes/dot (corresponding to 0.1/zg tubulin); line PBT lx: 0.1/.tg of pig brain tubulin/dot; lines PBT 5x, 10x, 20x: 0.5, I, 2/.tg of pig brain tubulirddot. The filters were incubated with all the antibodies cited above, and some of them were chosen to illustrate the whole. The antibodies were more diluted than above because we performed an ECL revelation which is far more sensitive than the revelation with NBT/BCIP. Lanes: 3, TAP 9311: 1:200; 5, TEU 435°: 1:100; 6, TEU 310°: 1:100; 10, TEU 4312: 1:50; 11, AXO 16: 1:300; 12, AXO 450: 1:300; 13, TAP 926G: 1:20; 15, 6-11B-l*: 1:500; 16, TEU 318°: 1:800; 18, TAP 931E: 1:20; 19, AXO 490: 1:20; 20, GT 335*: 1:7,500; 21, AXO 58: 1:2; 22, TAP 952°: 1:100. Each antibody has been simultaneously tested on tubulin denatured in electrophoresis sample buffer (illustrated here), on native tubulin
Tubulin diversity in Paramecium
®
101
Paramecium axonemes (AXO) lml
/
, 9 ~ - < ~ ~-
.
123
Sle__
4
5
6
7
8
g
9 10 11 12 13 14 15 16 17 18 19 20 21 22
-" ,ta
..
°
II
antisequence
others
CB
"Pig brain tubulin (PBT) 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
(X
t
"
..
©
3
5
6
10
11
O O
O
O
12
AXOlx
O
PBT Ix
•
•
PBT 5x
O
•
•
*
PBT 10x
O
t
•
O
PBT 20x
O
•
o
O
13
O
15
0
16
18
O
0
19
20
O 'O
•
t"
•
O
O.
•
•
•
•
O
•
O
•
21
O
22
O
I
and on protein markers in sample buffer. The dot staining was identical or slightly more intense with native tubulin, and negative or very weak with unrelated proteins. To express the reactivity of the antibodies with pig brain tubulin relative to Paramecium tubulin, we indicate the ratio of the amounts of Paramecium tubulin and pig brain tubulin yielding a similar staining. For example: in (3) with TAP 9311, the reactivity has been noted 1 because pig brain and Paramecium tubulins were equally reactive; in (5) the reactivity of TEU 435 ° has been noted < 1:20 because at least a 20 times higher amount of tubulin from pig brain relative to Paramecium was necessary to give a similar staining. These reactivity values, determined in this way for all antibodies, have been indicated in table II (dot reactivity).
102
A M C a l l e n et al
noblots of (cx + ~)-tubulins from Paramecium and porcine brain (fig 2 A, B, lanes 1-11); however, they yield a more diffuse upward staining than observed with the reference antibody D M 1 A which takes the form of a thin, sharp band, suggesting that D M 1 A may be recognizing only a subset of ~-tubulin (see Discussion). To delineate the location o f the epitope recognized by these antibodies, they were each reacted on peptide maps of Paramecium a-tubulin as well as against various segments of ciliate ~x-tubulins obtained as fusion proteins (which allow to cover the complete c~-tubulin sequence of ciliates except for the N-terminal first 22 amino-acids, as shown in figure 3). Figure 4A shows, as expected, that: i) the reference antibody D M 1 A whose epitope is located between residues 426 and 430 of vertebrate c~-tubulin [11] does not react with Euplotes fusion protein (E) which terminates at residue 412, but reacts with the C-terminal fragment (C) o f Stylonychia fusion protein; ii) antibody TU-01, which recognizes an epitope located in the segment 65-97 o f vertebrate cx-tubulin [34], interacts with Euplotes (E) and Stylonychia N-terminal fN) fusion proteins. Similarly to TU-01, all the antibodies in this section react conspicuously with Euplotes (E) and Stylonychia N-t~rminal (N) fusion proteins at e q u i v a l e n t
FUSIONPROTEINS
25
Euplotes (E)
1
Stylonychia ft.
- -(N)
22
MW
(c)
176 214
PARAMECIUM A X O N E M A L u - T U B U L I N P E P T I D E S
3O
L . - ~
1
I
25 21 19 15 13 I1 7 6.5 6
k__i.
~
~Jl . . . I
I . . . . . .
J
I I .a L - - .m .Jm-t. t. _ .
.........
JI--HA.
. . . . .
J
J
k. _ _ . L , - - I , . . . . L - [ll,.i,.
_I
- _s
t.. . . . .
I
~
....
i
C
a-TUBULIN
N
I
3 5 4 4 6 5 97 |1
I
II
1
214 226
I
I
426 4 3 0 449 II I U DMIA"
I
I
6 - I 1 B - I * TU-01*
C98"
100 i
159 i
281 i
Ac
Fig 3. Schematic localization of the Paramecium axonemal a- and ~-tubulin antigenic determinants described in this paper. The diagram represents: I) the structure of fusion proteins corresponding to fragments of a-tubulin genes of the ciliates Euplotes (E) and Stylonychia (N, C); 2) Paramecium axonemal a- and ~-tubulin peptides generated after proteolysis by V8 protease and reactive with the antibodies studied. The molecular masses (Mr) of the peptides are indicated (in kDa). The peptide positions within the sequence are listed in table lII. Dotted lines represent the intervals between their'extreme possible positions. The epitope positions of reference antibodies (*) are those determined on brain tubulin and are represented on the whole polypeptidic chains of Paramecium or- and ~tubulins and on their fragments by solid rectangles (a white rectangle means that the concerned antibody has not been tested). The epitope positions of the collection of antibodies studied here are inferred from their re.activities on fusion proteins and on Paramecium tubulin peptides and include the uncertainties in the positions of the smallest reactive peptides. The names of antibodies are those of table II. Ac designates the three anti-acetylated tubulin antibodies, aS designates 8 anti-cx-tubulin sequence antibodies (except AXO 16) and flS I anti-fl-tubulin sequence antibody (AXO 45°). All the data recapitulated in this figure are illustrated in the paper, except for those concerning antibody C 140.
412
l
TAP 956 98
136
254
i
i
i
449 i
o.S
AXO$8, TAP 952
PARAMECIUM I3-TUBULIN P E P ' r I D E S
30
1 ............
~
......... i._
21 11 6
L . . . . .
~
t....
,.I.i
. . . . . . . .
Ill
....
J
i
13-TUBULIN
N I i
j
i
153 165 II
241 256 I
C 140"
C
416 430 442
I
II
C 113" 188 !
D
I B*
310 i
TAP 956
257
442
I
I
~S: A X O 4S 442
257 l
l AXO
58, T A P
952
Fig 4. Immunoblotting analysis on fusion proteins. Coomassie blue staining of a gel loaded with fusion proteins: Euplotes (E), Stylonychia: C-terminal fragment (C), N-terminal fragment (N) (see fig 3), and Paramecium axonemes (A). A. Reactivity of anti u-tubuhn sequence antibodies. Dilution of antibodies: DM1A*: 1:1000; TU-01*: 1:500; TAP 931B: 1:10; TEU 435°: 1:50; TEU 310°: I:100; TEU 4312: 1:20. B. Reactivity of anti-acetylated ¢x-tubulin antibodies. Antibody dilutions: 6-11B-I*: 1:100; TEU 318°: 1:1000. Revelation was carried out using alkaline phosphatase-labeled secondary antibodies and NBT/BCIP; ECL revelation gave the same results (not shown). In A and B, the amounts of fusion proteins loaded on the gels were roughly equivalent to that of Paramecium tubulin (0.2/zg of axonemes corresponding to 0.1 #g of tubulin) as determined by gel scanning. This is visible by comparison of the stainings yielded by the reference anti-vertebrate tubulin antibodies on fusion proteins and axonemes. C. Compared reaetivities on eukaryote tubulin (Tub E: Euplotes tubulin; Tub S: Stylonychia tubulin) and on fusion tubulin (E, C, N) of antisequence antibodies (*) and of antibodies negative in our standard conditions (in A and B) on fusion proteins. Antibody dilutions: TEU 435*: 1:500; DM1A*: 1:15,000; TAP 956°: 1:500; AXO 58: 1:4. ECL revelation was used in order to increase the sensitivity of detection. Protein
Tubulin diversity in Paramecium
Coomassie
DMIA*
103
TUOI*
TAP 931B
TEU 435
TEU 310
TEU 4312
ECNA
ECNA
E C
ECNA
E C NA
m
i
__..~
E CNA
..........
6-11B-l*
N A
TEU 318
Eupi Styi Para (E) (C) (N) (A) I
!
Fusion proteins
®
| °
,"
! E NA TEU 435*
DM1A*
©
E C N A
TEU 435*
TAP 956
t
~
AXO 58
~41
mn,
It i
o
TubE
l/ a' -l -~ TubS
E
-
TubS
C
.--.o
D
TubE
'N
TubS
E
TubE
C
N
TubS
E
C
N
quantities have been increased relative to those used in A and B: Tub E and Tub S x 3 (relative to P a r a m e c i u m tubulin); E x 3, C x 28, N x 5 so that the fusion protein amounts are at least equivalent to those of eukaryote tubulins. All antibodies strongly reacted with Tub E and Tub S; neither T A P 956 ° nor A X O 58 reacted with E and N; AXO 58 did not react with C either, whereas TAP 956* weakly reacted with C: compare the equivalent labelings provided by D M I A * and TAP 956 ° on Tub S, and the dramatically different stainings yielded by both antibodies on C.
104
AM Callen et al
MW V8
13
I 3
I
2
1
0
I MW 0
1
2
3
i - - 50
50_ t. 1
t
35--
_ 34 _ 30
30_
_ F
I
21--
1
O
11--
I,D,
,al~ll
,i~1~
I i~1~-
9
10
_ 25 -- 24
21 __19 11
6
I
2
3
4
5
6
7
8
11
Fig 5. Silver stained peptide map of Paramecium axonemal a- and fl-tubulins, a- and fl-tubulins were separated and cut out from gels of 10/.tg axonemes performed at pH 8.25; it is assumed that tubulin represents 50% of the axoneme protein content• Lane 1, Rainbow molecular mass markers of 46, 30, 21.5, 14.5, 6.5 and 3.5 kDa; in the standard gel conditions used here and in figures 6A and 12 (resolving gel at pH 8.25), the 3.5-kDa marker is visible at or under the bromophenol blue migration front. Lanes 2 and 11 (MW), molecular masses (in kDa) of fl- and a-tubulins and their major immunoreactive peptides derived from proteolysis by V8 protease, determined from their electrophoretic migration (see table III). Lanes 6, 7, 2.5/.tg of 13- or a-tubulins respectively, denoted 0 in the upper line 'V8'. Lanes 5, 4, 3 and 8, 9, 10, peptide generated by proteolysis of fl- or a- tubulin with respectively 0.002, 0.02 and 0.2/.tg V8, named 1, 2, 3 respectively in the upper line 'V8'. For all peptide maps, the same denomination will be used to indicate the protease amount. Compare the peptide pattern of fl-tubulin, spread in the MW range extending from 35 to 6 kDa, contrasting with that of a-tubulin which is clustered in 4 MW zones: 35-30, 20, 11 and 6 kDa. Note, in lanes 3 and 10, the presence of a 30-kDa component which is certainly 'V8' protease itself [19].
amounts relative to P a r a m e c i u m axonemal tubulin. As examples, results obtained with monoclonal antibodies and hybridoma supematants TAP 931B, TEU 435 °, TEU 310 ° and TEU 4312 are shown in figure 4A. Thus, the epitopes recognized by all these antibodies must be located in a sequence of a-tubulin comprised between amino-acids 22 and 176. The results obtained by immunoblotting of peptide maps fit very nicely with the broad localization deduced from blotting on fusion peptides and allow to narrow the domain in which the epitopes are located. Figure 5 shows peptide maps o f P a r a m e c i u m axonemal a - and fl-tubulins performed at three V8 protease concentrations. In spite of different patterns, the similarities in molecular masses of main peptides originating from a - and fl-tubulins suggest that they derive f r o m c l e a v a g e s which o c c u r r e d in similar regions of both polypeptidic chains; this is confirmed by immunoblotting analysis (see figs 3, 6, 12). As an example,
the peptide map staining pattern yielded by the monoclonal antibody TEU 435 ° is illustrated in figure 6A. This antibody strongly reacts with a-tubulin fragments of 34, 30, 25, 24, 21, 15 and 11 kDa and, more weakly, on fragments of 13 and 7 kDa (lanes 5-7). No fl-tubulin peptide is recognized, as expected (lanes 2-4). The other anti-a-tubulin sequence ( a S ) antibodies react with the same peptides (another illustration is given with TEU 310 ° in figure 10). To d e t e r m i n e the l o c a t i o n o f these peptides in the sequence of a-tubulin, a variety of reference antibodies were used. Key results were obtained with antibody TU-01, (directed to epitope 65-97) which recognizes all these peptides (fig 6A, lanes 8-11) including the 7-kDa one (hardly visible on lane 8), but not the 11-kDa one, which is hence located on the right (towards the C-terminal end) of residues 65-97. The positions of all these peptides are represented in figure 3 and table HI. Since all aS antibodies react with the 11 and 7-kDa peptides together, the epitopes they
Tubulin diversity in Paramecium
recognize must be situated in the overlapping region of both peptides, beginning approximately downstream residue 97 and limited by the C-terminal end of the 7-kDa peptide. The latter is not recognized by 6-11B-1 (see below) and hence covers the region 45 (or 65) to 102 (or 136). This delimits the region 98-136, indicated as ' a S ' epitope on the schematic diagram in figure 3 and in table II. All the results described so far could suggest that all these antibodies are identical. However, the immunofluorescence patterns obtained on metazoan cells, 3T3 and PtK2 (data not shown), differ depending on the antibody (see table II). TAP 9311 and TAP 931B decorate the whole microtubule network as DM1A and DM1B; TEU 435 °, TEU 441 give a varying labeling depending on the fixation mode used (formaldehyde after Triton X-100 extraction or methanol) as other antisequence antibodies do; the other antibodies decorate 3T3 and PtK2 microtubule arrays either weakly or not at all. These observations are confirmed by immunoblotting tests over pig brain tubulin (fig 2B, lanes 3-11): when using the same amount of this tubulin as that of Paramecium, antibodies TAP 9311, TAP 931B and AXO 16 are found to be clearly positive over the ct-tubulin subunit and in these cases, the staining is sharp and without any smear. The other antibodies react either much more weakly (TEU 435 °) or not at all (table II). To better estimate the extent of cross-reaction of these anti-ciliate tubulin antibodies towards mammalian tubulin, dot blots using increasing amounts of antigen were carried out (fig 2C and table II). The most strongly reactive antibodies are again TAP 9311 and TAP 931B; TEU 435 °, AXO 16 and TELl 4312 yield a weakly positive response; all the others, TEU 310 °, TEU 441, TEU 4211G and TEU 4211H are negative. In summary, all the antibodies in this class appear to be directed against sequence determinants located in the N-terminal portion, probably 98-136, of t~-tubulin. However, the epitope(s) recognized by TAP 9311 and TAP 931B is (are) common to ciliates and metazoa; the epitope(s) recognized by TEU 435 °, TEU 4312 and AXO 16 would be only partly conserved in metazoan tx-tubulin, whereas the epitope(s) r e c o g n i z e d by T E U 310 ° , T E U 4211G, T E U 4 2 1 1 H seem(s) to be ciliate-specific.
Anti-~-tubulin sequence ([~S) antibody Although this antibody was not tested on E coli expressed ciliate fl-tubulin, the strong similarity of its immunodecoration pattern with reference antisequence antibodies and, conversely, the distinction with those provided by antibodies recognizing post-translational modifications suggest that AXO 45 ° is directed towards a sequence determinant. Indeed, in immunofluorescence tests over Paramecium and metazoan 3T3 or PtK2 ceils (data not shown), AXO 45 ° yields a pattern similar to those provided by the universal antibodies DM1A and DM1B. In agreement with the immunocytochemical observations, AXO 45 ° reacts conspicuously on pig brain fl-tubulin both on immunoblots and dot blots (fig 2B, C, lane 12). On immunoblots of Paramecium axonemes,,antibody AXO 45 ° reacts only over the fl-tubulin, yielding a sharp band (fig 2A, lane 12). On peptide maps (fig 6A, lanes 11-17), this antibody reacts over a 21-kDa fragment of fl-tubulin, also recognized by the reference antibody DM1B thus indicating that this peptide, containing the flS epitope, comprises the C-terminal domain of fl-tubulin (fig 3, tables II and III). It is remarkable that this peptide is weakly stained by both antibodies, and especially by
105
AXO 45 °. No peptide resulting from c~-tubulin digestion is reactive. In conclusion, the epitope recognized by AXO 45 ° is located in the C-terminal domain of fl-tubulin and common to ciliates and metazoa.
Antibodies reacting weakly or not at all with fusion proteins The category now analyzed corresponds to antibodies that fail to react distinctly on fusion proteins. As an example, TEU 318 ° is shown in figure 4B; when gels are loaded with the amounts of fusion proteins on which the anti-tx-tubulin sequence antibodies reacted (fig 4A), this antibody is negative on tx-tubulin fusion proteins, either from Stylonychia (C, N) or from Euplotes (E). This result can be due to two main causes: either the antibody is directed against a posttranslationaUy added determinant not occurring in E coli, or the constraints on the potentially reactive tubulin segment are such that, when associated to fl-gaiactosidase, it is not accessible or not in the proper immunologically recognizable conformation.
Antibodies probably directed against or- and fl-tubulin sequences: TAP 956 ° and TAP 926G These two antibodies have the same characteristics. In immunofluorescence tests, they display the same pattern as DM1A, DM1B, and our anti-sequence antibodies both on Paramecium and on metazoan 3T3 and PtK2 ceils (table II). On immunoblots, they label both the tx and fl bands of Paramecium axonemal tubulin with approximately equal intensity (fig 2A, lanes 13, 14); they also recognize cytoplasmic tubulin but the tx band is more strongly labeled than the fl one (fig 7A). These results fit well with the fact that they decorate all microtubule networks in Paramecium. On peptide maps of fl-tubulin (fig 6B, lanes 2-4 and 8-11), the same peptides of 34, 30, 11 and 6 kDa are labeled by TAP 956 ° (table lll) and by the reference antibody C 113 [5] which recognizes an epitope located at the level of amino-acids 241-256 of fl-tubulin. On peptide maps of axonemal a-tubulin (fig 6B, lanes 5-8 and 12-14), TAP 956 ° strongly labels peptides of 34, 30, 25, 24 and 6 kDa (table III), and weakly labels a peptide of 5 kDa, exactly in the same way as the reference antibody C 98 [5] which is directed against an epitope localized in the central domain of tx-tubulin, as the level of amino-acids 214 to 226. The two extreme possible positions for the smallest (6 kDa) t - and tx-tubulin peptides recognized by TAP 956 ° can be deduced from their respective reactivities with C 113 and C 98: these two antibodies permit to delimit the regions 188-310 in t - and 159-281 in ct-tubulins including the epitopes recognized by TAP 956 ° and TAP 926G (fig 3 and table ID. In agreement with the labeling of metazoan cells, they cross-react with pig brain tubulin: on immunoblots, both label the tx-subunit and TAP 956 ° also labels the fl-subunit very weakly (fig 2B, lanes 13, 14); on dot blots, an amount of pig brain tubulin ten times higher than that of Paramecium is required to obtain reactions of similar intensity (fig 2C, lane 13). In immunofluorescence tests, TAP 956 ° and TAP 926G react on the microtubules of Stylonychia (not shown). They are also readily positive on immunoblots of the tubulin extracted from Stylonychia and Euplotes ceils (fig 4C, TAP 956 °, Tub S, Tub E). However, they did not react with amounts of fusion proteins equivalent to those of positively reacting Paramecium axonemal tubulin (as in fig 4]3); if they did not react either when the amounts of Euplotes
106
AM Callen et al •i.
®
T E U 435 I I
V8
13
I
2
3
TU-01*
I
oc
1
I
2
I 3
I
tl
30 21.5_
2
1
0
- t
ql~
O
FL.--
aid
~
4
(9
5
6
~,
•
3
I
2
1
1
1
~ 15
-11 - 7
7
8
9
10
11 12 13 14 15 C 113"
=
1.
3
-21-
-.
I
13
2
-25
~
I
I
I
-30
T A P 956
V8
I
l '-3
-
3
l
I
I 13 I eCl
g-
6.5_
2
I
P
i
4 Odlb
14.5 . . . .
I
IMWI
i ;
46
¢.
A X O 45
ec
3
tn
2
I 3
C 98*
I
MW
16 17
I
I
13 3
I
I
2
1
ct 1
2
I 3
r'T.
46 - -
30
0"
|a
-50-
21.5 -
!
14.5 - -
o g
m
Q
- - 25 - -
41.
,Q _11__
.o
~
O
_ 6 _ g o
!
6.5
~q
- 34--- 30--
T
--
0"
3.5--
1
2
3
4
Fig 6. Immunoblotting of peptide maps of
5
6
7
8
9
10
11
12
13
14
Paramecium axonemal ct- and fl-tubulins with anti-tubulin sequence antibodies, a- and fl-tubulins came from gels of axonemes carded out either at pH 9.25, in A (lanes 2-10) and in B or at pH 8.25, in A (lanes 12-17). Peptides arose from proteolysis of 3.75/Jg fl- or a-tubulin by V8 protease (see fig 5 for denomination of protease amount) except in A, lanes 16 and 17, where each track loaded with 5/.tg was divided in two halves after transfer. A and B, lane 1, rainbow coloured protein markers with their molecular masses; in B, in order to get a good resolution of small peptides (see for example, the 3.5 kDa marker), particular gel conditions were used, involving double concentrations of Tris at pH 8.8 (see Materials a n d methods); lanes 11 and 8 (MW),
107
Tubulin diversity in Paramecium
fusion protein were tripled (fig 4C, E), they nevertheless reacted w e a k l y when the amounts o f Stylonychia fusion protein (214-445) were multiplied by 28 (fig 4C, C). In conclusion, these data suggest that both antibodies are directed to a sequence epitope (see Discussion) present in the central domains o f ~z- and ~ t u b u l i n s (159-281 and 188-310, respectively), and common to ciliates and metazoa.
Antibodies certainly or presumably directed against posttranslational epitopes: TEU 318 °, TEU 348 °, TAP 931E, TAP 952 °, AXO 58, AXO 49 ° A large set o f antibodies was first assigned to this category on the basis o f i m m u n o c y t o c h e m i c a l tests: most o f these antibodies yield decoration patterns very similar to those obtained using reference antibodies directed against known p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s . T w o a d d i t i o n a l criteria were the absence o f immunoreactivity with E coli synthesized fusion peptides even at very high protein amounts (as an example, A X O 58 is shown in fig 4C) and, for one o f them ( A X O 49°), the delay in appearance o f immunoreactivity on assembled microtubules (see Discussion). These various antibodies could be subdivided into four clearly distinct groups, the first one directed against acetylated tubulin, and the last three assumed to be against other post-translational modifications.
Antibodies directed against acetylated ~x-tubulin (Ac antibodies). A s s i g n m e n t o f a n t i b o d i e s to this group h e a v i l y relied on the existence o f a very well characterized antibody directed against acetylated lysine 40 o f cz-tubulin, 6-11B-1 [44]. Three anti-Paramecium tubulin antibodies belong to this group: T E U 318 °, T E U 348 ° and T A P 931E, and their similarity with 6-11B-1 is striking in many respects. In immunofluorescence tests on Paramecium, they decorate cold or nocodazole stable microtubules and only the tip o f cilia (fig 8a) exactly as 6-11B-1 [2, 15, 70]. On immunoblots of Paramecium axonemal tubulin, they label only the cz-tubulin band, with a smear extending all the way to the position o f ~ t u b u l i n (fig 2A, lanes 15-18); in contrast to the strong staining yielded on axonemal tubulin, only a very small amount o f cytoplasmic tubulin migrating at the level
Table III. Estimated molecular masses and sequence positions of proteolytic fragments of Paramecium axonemai ~z- and ~tubulins reactive with the antibodies studied here. The molecular masses of the peptides were determined from plots of log M r vs migration after SDS-PAGE; mean values ± 2 standard deviations Listed below give a 95% confidence interval. The number of residues were calculated using 115 as mean molecular mass per residue. The peptide positions were inferred from their antigenic reactivities with reference antibodies, and the two extreme positions of their N- and C-terminal ends compatible with their reactivities were indicated, taking into account the experimental errors.
Apparent molecular mass (kDa) (daltons)
~x-tubulin peptides
,/)-tubulin peptides
34 30 25 24 21 19 15 13 11 7 6.5 6 34 30 21 11 6
33700 + 2200 30300 ± 2600 25100 ± 1400 23900 -+ 1400 20600_+ 1200 19200± 1200 14700 ± 2 800 12600 ± 3600 10700 ± 3000 6800 ± 1500 6500 ± 1100 6000 :e 1800 34200 ± 2200 2 9 7 0 0 ± 1800 20600 _+2000 11300±2000 6000±2000
Apparent number extreme of residues sequence positions N C 293 ± 13 263 ± 23 218 ± 12 208 ± 12 179 ± 10 167 ± 10 128 ± 24 110 ± 31 93 ± 26 59 ± 13 56 ± 10 52±13 297 ± 19 258 ± 16 179 + 17 98 ± 17 52 ± 17
1-35 1-35 1-35 7-35 1-35 254-293 1-65 1-35 98-147 45-65 1-35 159-214 1-138 1-153 257-281 166-241 188-241
274-346 240-320 226-264 226-254 169-213 430 ~ 9 104-213 102-175 164-213 102-136 46-100 226-281 278--415 256--415 430--442 256-355 256-310
o f the a x o n e m a l to-band reacts with antibodies 6-11B-1, T E U 318 ° and T E U 348 ° (fig 7B). These data validate the immunofluorescence ones showing a lack o f decoration o f c y t o p l a s m i c m i c r o t u b u l e s by these antibodies, h o w e v e r , .they do not account for the weak decoration o f the cilia.
molecular masses of cz- and ~-tubulins and their major immunoreactive peptides. In A, immunoblotting was carried out with TEU 435 ° (1:103) (lanes 2-7), TU-01* (1:5.103) (lanes 8-10), AXO 45 ° (1:2.5) (lanes 12-15), with respectively DMIB* (1:103) and AXO 45 ° (1:5) on each half-track of lane 16, and with respectively AXO 45 ° (1:5) and DM1A* (1:2.104) on each half-track of lane 17. It must be stressed that when ~tubulin bands were cut out from gels made at pH 9.25, in which/~-migrates faster than ot-tubulin, no staining of peptides was visible with anti-cz-tubulin sequence antibodies, even at long exposure times using chemiluminescence. In contrast, when ~tubulin was cut out from gels made at pH 8.25, in which the ~ b a n d is above the ~ one, we noticed a staining of peptides at the level of those deriving from a-tubulin. This can be attributed to contamination by ~z-tubulin on the primary gel, resulting from the slower migration of some forms of cz-tubulin which yield smeared bands extending all the way to the position of ~-tubulin on gels and blots (fig 2A, CB and lanes 3-11). Remark in lanes 16 and 17 the strong staining of the 19-kDa ~z-peptide afforded by DMIA* in comparison with the weaker staining of the 21-kDa/3-peptide yielded by DM1B*, used 20 times more concentrated. It seems that the lower amount of the latter peptide, visible on a silver stained gel (compare in fig 5 the staining of the 21-kDa ~peptide in lane 5 to that of the 19-kDa ~x-peptide in lane 8) cannot completely account for this difference. Additionally, with AXO 45 °, a still weaker staining of the ~peptide is exhibited (see Discussion), and no staining at all of the cz-peptide. In B, immunoreactivity with TAP 956 ° (1:10) in lanes 2-7, with C 113" (1/.tg/ml) in lanes 9-11, and with C 98* (0.6/ag/ml) in lanes 12-14. The same whole filter has been first treated with TAP 956* (monoclonai) then with a biotinylated anti-mouse IgG, and further processed as described in Materials and methods, yielding the staining observed on both ~z- and ~peptides in lanes 2-7; secondly, with C 98* (polyclonal) then with a biotinylated anti-rabbit IgG, producing the staining of a-peptides only, seen in lanes 12-14; thirdly, with C 113" (polyclonai), resulting in the labeling of/J-peptides solely, visible in lanes 9-11. The same results had been obtained after single staining of ~z- and ]3-peptides with the three antibodies, and in particular, C 98* did not label/~-peptides whereas C 113" did not stain the cr ones. Note in lane 6, the presence of a peptide stained by TAP 956 ° at the level of the/~ 11-kDa peptide and not labeled by C 98*; it therefore appears to result from a contamination of ~z- by ~tubulin since the tubulin bands were cut from gels of axonemes made at pH 9.25, in which some forms of ~-tubulin migrate slower and are spread until the level of ~z-tubulin. Conversely, this staining is not seen when the tubulin bands are cut from gels made at pH 8.25 where (x-migrates faster than/3-tubulin.
108
A M C a l l e n et al
I
, ct ,
4
,i
i
i
" "! i=-la la -
II -
[ i [
Axo Cyt
Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt Axo Cyt I II II I
A
B
C
Anti-acetylated tubulin antibodies Fig 7. Comparative immunoblotting analysis of Paramecium axonemes (Axo) and cytoplasmic (Cyt) extract. Relative reactivities of antibodies which label weakly (A) or not at all (B, C) fusion proteins. Protein mounts: axonemes, 0.3 gg; the cytoplasmic extract contained an equivalent amount of tubulin (see the relative staining of DM1A* on Axo and Cyt). ECL revelation was carded out. A. Presumed anti-tubulin sequence antibodies: TAP 926G: 1:500; TAP 956°: 1:103. Both ix- and fl-tubulins are labeled. B. Anti-acetylated tubulin antibodies: 6liB-l*: 1:500, TEU 318°: 1:104, TELl 348°: 1:2.10 3, TAP 931E: 1:600. Only tx-tubulin is stained, but very weakly in cytoplasmic extract` C. Antibodies directed against putative post-translational modifications different from acetylation: GT 335*: 1:105, AXO 58:1:103, TAP 952°: 1:4.104. GT 335* reveals on axonemes a doublet the faster component of which migrates like fl-tubulin and is also stained by AXO 58 (which reacts with additional bands); on the cytoplasmic extract, reactivity is seen at the level of fl-tubulin, as with AXO 58; high moleeul~ mass polypeptides are also visible, as noted by Br6 et al [10]. TAP 952 ° labels both fl- and tx-tubulins.
On peptide maps of t~-tubulin, the four Ac antibodies strongly react over the same N-terminal peptides of 34, 30, 25, 24 and 21 kDa and faintly on lower molecular mass peptides (fig 3; fig 9, lanes 3-9; table IT/). No fl peptide is stained (fig 9, lanes 1-2). In order to narrow the localization of the epitopes recognized by these Ac antibodies, a comparison of their reactivities with those of the anti-ct-tubulin sequence (tzS) ones was carried out focussing on the low molecular mass peptides (fig 10). The smallest peptide well recognized by both categories of antibodies is a 13-kDa one, whereas the smallest peptide reactive with all Ac antibodies is one of 6.5 kDa. The latter peptide differs from the 7-kDa one reactive with the aS antibodies; this 6.5 kDa peptide permits to delimit the region 1-100, indicated as 'Ac' epitope on the diagram in figure 3 (see also tables II
and 1II). As 6-11B-l, the Ae antibodies are fully negative on all types of tz-tubulin fusion proteins (as an example, TEU 318 ° is shown in figure 4B) even if amounts of proteins are very high. Immunoblotting tests over pig brain tubulin yield a sharply decorated band, without smear, of variable intensity with the different antibodies (fig 2B, lanes 15-18). This is confirmed by dot blots: TAP 931E reacts on pig brain tubu-
lin only when a 10 times higher amount is deposited with respect to Paramecium axonemal tubulin; with TEU 318 ° and TEU 348 °, a five-fold ratio is sufficient to obtain equivalent intensity while 6-11B-1 is intermediate (fig 2C, lanes 15, 16, 18). As 6-11B-1, the three antibodies fail to decorate microtubules in PtK2 cells, the tubulin of which is non-aeetylated [54] while they decorate the stable microtubule network in 3T3 cells (not shown). In the latter cells, the primary cilium, the midbody and the mitotic spindle are also strongly decorated. To definitely establish that our antibodies are indeed directed against acetylated tz-tubulin, we performed chemical acetylation on native purified pig brain tubulin. As already observed by Piperno and Fuller [53], acetylated tubulins, o~ Ac and fl Ac, migrate slower than their native counterparts, tz and fl (fig 11A: Coomassie blue and blots with DM1A and DMIB). This slower migration is very likely due to decreased SDS binding resulting from neutralization of basic lysine residues [16]. It can serve as a 'signature' of tubulin acetylation. Note that native porcine brain tubulin contains a minor amount of acetylated tubulin migrating slighty higher than the major native t~-tubulin revealed by DM1A (fig l l B ) , whereas a strong reaction with the four antibodies is visible on in vitro fully acety-
Tubulin diversity in Paramecium
109
:,
b ~''~
',' /
.~
,,
i','~ /
'
.~ \,
Fig 8. Projections of Paramecium cells decorated with the following antibodies: (a) TEU 348 ° (1:10), (b) TAP 952 ° (1:20), (e) AXO 58 (1:2) and (d) AXO 49 ° (1:5) observed by confocahmicroscopy. Each antibody yields a specific staining of the cilia: only the tips (arrowheads in a) are strongly decorated with TEU 348 °, while, in addition, the rest of the axonemal structure is decorated along its whole length with TAP 952 ° (b) or only along its proximal part with AXO 58 (c) including the tips (arrowheads in b and e); in contrast, the strong decoration of axonemes by AXO 49 ° (d) contrasts with that yielded by the other antibodies, a. This projection collecting all the information from inside the cell shows that only some microtubules are decorated with the TEU 348° antibody: subpellicular microtubules associated with the anterior left area of the cell (P), postoral fiber (Po) and cytopharyngeal microtubules (Cf) associated with the oral apparatus (O), microtubules associated with the contractile vacuole system (V). In contrast, the TAP 952* antibody Co) also decorates the internal meshwork of microtubules (arrows). AXO 58 (e) yields a pattern similar to TEU 348 °, while AXO 49 ° (d) is more restrictive: it decorates only the postoral fiber (Po) associated with the oral area (O) and a short portion of the microtubules associated with the contractile vacuole system ((V), partly out of the focus). Scales are 254//m.
1 10
AM Callen
T E U 318
I I
I
V8
3
6-11B-I*
I I
a
1
1
et al
2
3
I I
a 3
2
MW
1
'
MW -30 -25
R
~ ~
-45 -34
-21
30 ~
-15
-13 -11
- 25 -21
-7
--10
1
2
3
4
5
678
9
Fig 9. Immunoblotting of peptide maps of Paramecium axonemal a- and fl-tubulins with anti-acetylated tubulin antibodies. Here, tubulins were separated from a gel of axonemes at pH 9.25 in order to avoid contamination of 13- by a-tubulin. Peptides arose from digestion of 3.75/Jg fl- or cx-tubulin by V8 protease (see figure 5 for protease amounts). Immunoreactivity with TEU 318 ° (1:200) (lanes 1-5) and with 6-11B-I* (1:50) (lanes 6-8). Lane 9 (MW), molecular masses (in kDa) of major immunoreactive peptides.
lated a-tubulin, together with a large migration shift. N o reaction is detectable on fl-tubulin in all cases (best illustrated with 6-11 B- 1 and T A P 931E in fig 11B). In conclusion, these three new anti-tubulin antibodies are clearly directed against an N-terminal acetylated epitope o f a-tubulin. T A P 9 5 2 °. T h i s a n t i b o d y a t t r a c t e d a t t e n t i o n b e c a u s e it a p p e a r e d to d e c o r a t e both the c i l i a along their w h o l e lenght and nearly all the i n t r a c e l l u l a r m i c r o t u b u l e n e t w o r k s o f P a r a m e c i u m (fig 8b). W h e n T A P 952 ° is c o n s i d e r a b l y diluted, only the tip o f cilia is l a b e l e d and w h e n l e s s d i l u t e d , a g r a d i e n t o f l a b e l i n g is o b s e r v e d d e c r e a s i n g f r o m the p r o x i m a l to the d i s t a l p o r t i o n o f cilia, as has been observed, albeit less clearly, with A X O
Antibody
58 (fig 8c). On i m m u n o b l o t s obtained from gels m a d e at pH 8.2,
1 2 Fig 10. Compared immunoblotting of a peptide map of Paramecium a-tubulin with an anti-acetylated tubulin antibody (lane 1) and an anti-a-tubulin sequence antibody (lane 2). Peptides were generated by proteolysis of 5 pg o~-tubulin by 0.2/.tg V8 protease and separated in standard gel conditions at pH 8.25; after transfer, one track was cut in two halves; one half-track (1) was incubated with 6-11B-l* ascitic fluid (1:103) and the other (2) with TEU 310 ° (1:200). Note the good visualization of minor peptides achieved here (in contrast to fig 9) by using high protein loadings and high antibody concentrations (6-11B-1" ascitic fluid instead of culture supernatant) and the resolution of the 7 and 6.5 kDa bands. Our anti-acetylated tubulin antibodies stained the same peptides as 6-11B-I*.
T A P 952 ° reacts with both o~- and fl-axonemal tubulins. The bands are sharp and without smear (fig 2A, lane 22). The antibody also reacts with P a r a m e c i u m cytoplasmic tubulin, the staining at the level o f fl being stronger than at the level o f o~-tubulin (fig 7 C). These results fit well with the labeling of both cytoplasmic and axonemal microtubules observed by immunofluorescence. On i m m u n o b l o t s o f p e p t i d e m a p s , T A P 952 ° r e a c t s with axonemal peptides o f 21 k D a from fl-tubulin and o f 19 k D a f r o m ~ - t u b u l i n , a l s o l a b e l e d b y D M 1 B a n d D M 1 A , respectively (fig 12, lanes 11-20), and therefore situated in the C-terminal d o m a i n o f each subunit (fig 3 and tables II, III).
Fig 11. Demonstration of anti-acetylated a-tubulin antibodies by means of in vitro acetylation of pig brain tubulin. Long gels (12 cm) were used to resolve acetylated co- and ]J-tubulin bands. Tubulin amounts: Coomassie blue: 0.8 pg/well before (C) and after acetylation (Ac); blots: 0.4 pg/well. Antibody .dilutions: DMIA*: 1:5.104, DM1B*: 1:5.104, 6-11B-I*: 1:5.102, TEU 318°: 1:4.104, TAP 931E: 1:50, TEU 348°: 1:5.103. ECL revelation was carried out. A. Coomassie blue: note the slower migration rates of a- and fl-tubulins after acetylation: compare a Ac with ~, fl Ac with ft. DM1A* and DMIB* label respectively ct- and fl-tubulin before (C) and after acetylation (Ac); migration shifts are visible on blots as seen on the gels. The level of the bands stained by DMIA* is slightly higher than the positions of the other bands because they correspond to tracks situated on the left side of the gel. 6-11B-l*, TEU 318 °, TAP 931E, TEU 348 ° label strongly a-tubulin after acetylation whereas they yielded a very weak staining on native pig brain tubulin. B. In order to get a
Tubulin diversity in Paramecium
®
l 11
Coomassie Blue
Or,--
•
B
0¢ Ac -Ac
"
-
,
-.
m
,q
f--
-m
.me
.-,D
C Ac
C Ac
C Ac
-
Ac
C
C Ac
,
®
C Ac
,
.
C Ac
j
¢xAc Gt
d - - 13Ac
I
II
before acftylation (C)
after acetylation (Ac)
better separation of ¢x- and ~-tubulins, SDS from Sigma has replaced SDS from BDH [8] in the gel and electrophoresis buffers. After transfer, each track has been cut in two halves and treated by two different antibodies. Before acetylation, 6-11B-1" labels weakly a minor form of a-tubulin not stained by DMIA* and which migrates slightly slower than the major form labeled by DM1A*. After acetylation, 6-11B-I* and TAP 931E label strongly a thick band at the level of that stained by DMIA* (c~ Ac) and faintly a band situated lower, also visible in A. The labeling of the acetylated ]3-tubulin by DMIB* (~1Ac) at a still lower level shows unambiguously that the faint band does not correspond to acetylated ~-tubulin.
112
AM Callen et al
AXO 58
I I
II
1
II
I
ll31O IMwl
I 2
V8
TAP 952
.<
0
0
1
2
3
1
1
1
0
~.
II
I
13 2
~
I
I 131 0
1
2
3
1 IW
30
1 r
•
OQ
46_
I
-
21.5-
°
~'
,21, "=- 19-
0,~
QB,
°
='QD
14.5 6.5_
1
2
3
4
5
6
7
8
9 10
11 12 13 14 15 16 17 18 19 20
Fig 12. Immunoblotting of peptide maps of Paramecium axonemal ¢x- and ~tubulins with presumed anti-tubulin post-translational modification antibodies. Here, tubulins were separated from a gel of axonemes at pH 8.25. Peptides were yielded from proteolysis by V8 protease of 3.75 #g/3- or ¢x-tubulin (lanes 2-3, 6-8), 5/Jg (lane 9), 2.5 #g (lanes 10, 12-13, 16-20). Undigested/3- and a-tubulin amounts were 3.75/tg (lanes 4-5) and 1/.tg (lanes 14-15). In lanes 9, 10, 19, 20, each track was cut after transfer in two half-tracks. Lane 1, protein markers and their molecular masses (in kDa). 11 (MW), molecular masses of undigested a- and ~-tubulins and their immunoreactive peptides. Immunoreactivity with AXO 58 (undiluted) in lanes 2-8 and on half-tracks of lanes 9 and 10; TAP 952 ° (1:20) in lanes 12-18 and (1:50) on half-tracks of lanes 19 and 20; DMIB* (1:103) and (1:5.103) on half-tracks of lanes 9 and 19, respectively; DM1A* (1:2.104) and (1:104) on half-tracks of lanes 10 and 20, respectively. Remark in lanes 9 and 10 that the equivalent staining of the 21-kDa ~]-peptide with DM1B* and DM1A* on one hand, and with AXO 58 on the other hand, is due to the different quantities o f ~ and a-tubulins (5/zg versus 2.5/zg). When an equivalent low tubulin amount (2.5/Jg) is loaded as in lanes 19 and 20, the labeling of the 21-kDa/~-peptide with DM1B* and AXO 58 is very faint. Note also the presence of an additional fl-peptide of ~ 19 kDa, labeled by DM1B* and only visible at high protein levels (compare lane 9 here and 16 of figure 6A with lane 19 of this figure). Note in lanes 19 and 20 the stronger staining of the 19-kDa ~x-peptide compared to that of the 21-kDa/ff-peptide by TAP 952 °, which could be probably correlated with the lower amount of the latter peptide.
T A P 952 ° does not react with fusion proteins (not shown). It fails to react with pig brain tubulin in immunoblotting and with c y t o p l a s m i c m a m m a l i a n tubulins in immunofluorescence tests (fig 2B, C, lane 22 and table II). This antibody therefore appears to be ciliate-specific, at least as concerns the cytoplasmic microtubules (note however that the cilia of metazoans have not been tested). A X O 58, an antibody yielding a pattern similar to G T 335, an anti-glutamylated tubulin antibody. A X O 58 was not successfially sub-cloned. However, we present some of its characteristics which are unique while others are reminiscent of GT 335 antibody which is known to react with polyglutamate side chains located in the C-terminal part of tubulin [75]. In immunofluorescence tests on Paramecium cells, AXO 58 decorates cilia in both the proximal and distal domains, as well as a stable population of cortical and cytoplasmic microtubules (fig 8c). This is very similar but not identical to the pattern provided by antibody GT 335 which, in addition, labels a sub-population of dynamic mierotubules [10].
On immunoblots obtained from gels of Paramecium axonemes made at pH 8.2 (fig 2A, lanes 20, 21), AXO 58 labels a band which migrates at the level of ~-tubulin, as was observed with GT 335, but also a band at the level of a-tubulin not recognized by GT 335 (fig 7C). Conversely, GT 335 stains a band which migrates just above ~tubulin, apparently not recognized by AXO 58. Thus, both antibodies seem to react with different forms of axonemal tubulin, which display different apparent molecular masses on SDS gels. Finally, as GT 335, AXO 58 reacts with cytoplasmic tubulin, at the level of the fl subunit (fig 7C). On immunoblots of peptide maps, AXO 58 mainly reacts with 21-kDa and 19-kDa peptides respectively originating from axonemal ~ and ¢x-tubulins, and respectively also labeled by D M I B and DM1A (fig 12, lanes 2-11), indicating that they are both located in the C-terminal part of each subunit (fig 3 and tables II, IlI). This result is somewhat different from that obtained with GT 335, which labeled, after tubulin proteolysis, peptides displaying shifts in migration relative to those of the 21- and 19-kDa ~- and o~-peptides
Tubulin diversity in Paramecium
!f
1 13
\
,j
I:1
,
Fig 13. Two projections of Paramecium cells at approximately the same stage of division and decorated with (a) TEU 348 ° (1:10) and (b) AXO 49 ° (1:5) antibodies. In addition to the oral apparatus (O) and the cilia, only microtubular rootlets associated with the two contractile vacuole systems (V) are decorated by AXO 49 ° (b). The latter correspond to parental systems (Vp), decorated by both antibodies (a, b), while the newly assembled ones (Vn in a) are not decorated by AXO 49 ° (b). In addition, transient structures, such as the elongation spindles of the micronuclei (e) or the microtubules located inside the macronucleus (ma), visible in (a) are again not decorated by AXO 49 ° (b). Scales are 25 #m.
(Levilliers et al, in preparation), again suggesting that different forms of tubulins would be recognized by both antibodies. Since/3-tubulin fusion proteins were not available, only the test against cx-tubulin fusion peptides could be carried out and proved to be fully negative, even when using high levels of fusion peptides, containing amounts of tubulin at least equivalent to that of the corresponding eukaryote tubulins on which the AXO 58 antibody reacts (in fig 4C, A X O 58, compare E, C, N to Tub E, Tub S). AXO 58 fails to react both on microtubules of PtK2 and 3T3 cells in immunofluorescence tests (table II) and on pig brain tubulin in immunoblots and dot blots (fig 2B, C, lane 21). This strongly contrasts with the distinct reactivity of GT 335 on PtK2 and 3T3 cells (not shown) and on pig brain tubulin both in immunoblots and dot blots (fig 2 B, C, lane 20).
AXO 49 °, an antibody directed against a post-translational modification similar to that recognized by a polyclonal antibody. This antibody is one of the few monoclonals which decorates strongly, in Paramecium, the cilia along all their length, except their tip, along with a restricted subpopulation of very stable microtubules (fig 8d). Its immunocytochemical labeling pattern is very similar to that yielded by a rabbit polyclonal serum raised against axonemal tubulin of Paramecium [14]. In addition, as in the case of the polyclonal serum [2] the reactivity of A X O 49 ° is detected over microtubules only
following a substantial lag after their assembly. For example, figure 13 shows that the newly assembled contractile vacuole system is not labeled by A X O 49 °, while it is decorated by TEU 348 °, an anti-acetylated tubulin antibody. On immunoblots of Paramecium axonemes, A X O 49 ° reacts with both ~x- and ]~-axonemal tubulins (fig 2A, lane 19). In contrast, it is fully negative over cytoplasmic Paramecium tubulin (not shown), in agreement with the immunocytochemical data. Moreover, this antibody is fully negative on cytoplasmic metazoan tubulin as shown by immunofluorescence tests over 3T3 and PtK2 cells (table II) as well as on immunoblots and dot blots over pig brain tubulin (fig 2B, C, lane 19). These data, combined with the fact that A X O 49 ° does not react over fusion proteins, strongly suggest that this antibody recognizes a post-translational epitope; it will be characterized in detail in a forthcoming paper (Levilliers et al, in preparation).
Discussion At the start of the present work, we had set ourselves the aim of isolating an array of monoclonal antibodies to ciliate tubulin as broad and diverse as possible. This is why we introduced variations in the type and mode of presentation of the tubulin antigen. Out of three fusions, a large diversity of antibodies was obtained, including both sequence and
114
AM Callen et al
post-translational modification-directed ones and, in particular, among the latter, a set of four antibodies, three of which correspond to new specificities previously undescribed in the literature. We will analyze the strength of these conclusions, evaluating the level of certainty with which both the nature and the location of epitopes has been established.
cut from the same track (see fig 6A, lane 16, fig I0 and fig 12, lanes 9-10, 19-20). This can be confirmed by double staining with both antibodies but one cannot exclude a comigrafion of two different peptides which would account for the simultaneous staining of a band by two antibodies. The well identified anti-o~-tubulin sequence antibodies and the location of their epitope(s)
Methodological considerations on peptide maps
The location of the epitopes has been determined through immunoblotting of peptide maps generated by various concentrations of V8 protease, yielding an array of peptides ranging from large molecular masses (30-20 kDa) to small ones (5 kDa). One preliminary point related to the structure of the protein under digestion and several comments on the technical advantages and some pitfalls of the method used can be made. The cleavage sites generated by V8 protease on SDSmercaptoethanol-treated Paramecium tubulin in acrylamide we inferred from the peptide sizes are situated in regions 100-200 and ~250--300 of ~x- and/$-tubulins also found by Serrano et al [63] and De la Vina et al [17] after cleavage of native soluble bovine brain tubulin by various proteases. This suggests that the so-called 'denatured' tubulin such as used in the 'Cleveland' procedure would not be completely unfolded and would keep a certain degree of its initial three-dimensional structure as compatible with 3 proteolytitally defined large compact regions [17] consisting of the N-terminal, middle and C-terminal thirds of the molecule: these regions presumably correspond to real structural features of native tubulin. The simultaneous separation of high and low molecular mass peptides on a single gel was rendered possible by the use of a 15-20% acrylamide gradient, which is not common for peptide map blotting. For most peptide maps presented here, involving a lower gel at pH 8.25, peptides of 6.5 kDa were well resolved (see figs 6A and 10), whereas, by using double "Iris concentrations both in the lower gel buffer (adjusted at pH 8.8) and the running buffer, peptides of 3.5 kDa could be resolved (compare the M r markers of fig 6A and B). Finally, the use of ECL revelation for immunoblots permired to visualize peptide staining otherwise not detected by classical coloured revelations (using NBT/BCIP or diaminobenzidine). This was the case for the small peptides of 11 and 7 kDa recognized by the anti
When a monoclonal antibody remains reactive over a portion of tubulin synthesized in E coli, we conclude that the antibody is directed to a sequential epitope of tubulin. This is corroborated by two 'internal controls': reference antibodies known to be directed to a primary sequence determinant such as TU-01 or DM1A indeed remain positive over fusion proteins and, conversely, 6-11B-1, an antibody whose reactivity is known to depend on the post-translational acetylation of lysine 40 [44] indeed completely fails to react over fusion proteins. In addition, all our antibodies that are positive on fusion proteins yield on Paramecium cells immunoeytochemical decoration patterns similar to those of reference anti-sequence antibodies and quite different from those of reference anti-modification ones. On the basis of these arguments, nine of our antibodies have been classified as being directed against an a-tubulin amino-acid sequence in the broad sense, the contiguity of which would result either from the primary structure or from the polypeptidic chain folding. The location of the epitope(s) has been further narrowed by peptide map blotting. For eight of the anti-cz-sequence (o~S) antibodies, the epitope appears to be located between positions 98 and 136 as deduced from the two smallest peptides (of 11 and 7 kDa) which they recognize (fig 3 and tables II, 131); however, taking in account the uncertainties in molecular mass and peptide position determinations discussed above leads to a conservative estimate of 150 for the extremity of the 7-kDa peptide. The region 98-150 of Paramecium cz-tubulin therefore appears to be specially immunogenic, in agreement with predictions of antigenic determinants of brain tubulin based on amino-acid hydrophilicity and with the fact that it corresponds to a zone of preferential cleavage by proteases [17, 49], hence probably accessible and immunogenic. In fact, it is also in the region 90-160 that Wolff et al [74] locate the epitope of the monoclonal antibody 111 B52 C2 directed against pig brain cx-tubulin. Moreover, this antibody seems to label the same peptides of Paramecium axonemal tubulin as our anti-sequence antibodies (not shown). In fact, our eight antibodies are not identical: one (TAP 931B) belongs to the IgG3 class while all the others belong to the IgG1 one; moreover, their reactivities against pig brain tubulin differ. We have tried to identify amino-acid substitutions distinguishing ciliates from mammals: they could.be immunogenic when ciliate tubulin is injected into a mouse and could belong to the epitopes of the ciliate-specific aS antibodies, ie at least TEU 310 °, TEU 4211G, TEU 4211H. When conservative substitutions are discarded, five amino-acid differences shared by all ciliate cx-tubulin sequences examined (12 belonging to nine species) are identified in the region 98-150: Cys/Val at position 118, AsrdGln at position 128, Val/Phe at position 141, Leu/Phe at 149 followed by Gly/Thr at position 150 (fig 14A). As, all the antibodies of this category are strictly cz-tubulin specific, it may be inferred that the epitope(s) towards which they are directed is (are) absent in the ~chain. Comparing sequences of both ~- and/~-polypeptidic chains of Paramecium tubulin in this region (fig 14B), it can be seen that they are identical
Tubulin diversity in Paramecium
Parameczum
91 QLISGKEDAA
Mammal
---t ........
Paramecium ct M~--.~ 1
Paramecium
NNFARGHYTI y .......
RIRKLADNCT
GLQGFLVFHS
Q
VGGGTGSGLG F . . . . . . . FT
151 Sr.7.7.~.RLSVD Y G K K S K L G F T I Y P S P Q V S T A .m. ~--S - - - A
VVEPYNSILS THSLLEHTDV t --tT---s-C
CVMLDNEAIY A F - v ......
211 DICRRNLDIE
SLRFDGALNV
270 PYPRIHFMLC .P-A
RPTYTNLNRL
Mamm~ i
®
GKEIVDLCLD .... i - - V - -
115
IAQVISSLTA S-iv--i
91
DITEFQTNLV 1
4,4,
4,
Paramecium QLISGKEDAA Paramecium NFvF-QTG-G
NNFABEHYTI GKEIVDLCLD --W-k .... E - A - I i - S V - -
RIRKLADNCT Vv--E-eG-D
89
Paramecium
151 ST.T.T.r.RL~. Y G K K S K L G F T
P a , r a m e e i mn t - - i S k v R E e - P D r I M E T - s
IYPSPQVSTA
I S I L 5
iV---K--DT
~ k T - -
IA0VISSLTA vSAAm-Gv-C
SLRFDGALNV C---~-Q--S
THSI~HTDV V-Q-v-NA-E
149
211 Paramecium ~_T_~E Paramecium
270
RPTYTNL~ T T---GD--H-
209
268
Fig 14. Partial amino-acid sequence comparison of tubulins. A. Paramecium and mammalian consensus a-tubulin sequences. The arrows point the positions of non-conservative substitutions between residues 98 and 150. The lower-case letters indicate the conservative substitutions. B. a- and ~tubulins from Paramecium. A gap was introduced in ~tubulin in order to obtain the best alignment. The numbering of residues is therefore different between a- and ~- tubulins. Negative types indicate homologous regions between a- and ~- tubulins at the potentially immunogenic sites (see Discussion). The homologous regions u(272-277), ~(270-275) have not been displayed because they contain one non-conservative substitution. Paramecium a- and ~-tubulin sequences are from P Dupuis [20, 21]; mammalian u-tubulin consensus sequence (from 14 sequences belonging to five mammalian species) and alignments were compiled by H Philippe (personal communication).
from residues 138 to 150, except at two positions where conservative substitutions are found: at 141, Val/Leu and at 149, Leu/Met; the sites at positions 141, 149 and 150 are thus excluded. At positions 118 and 128, amino-acid differences are found between the a and fl chains: Cys/Val including a polarity change and Asn/Gly without any polarity change; they remain as potential candidates for the antigenic determinants of the ciliate-specific o~ antibodies. Finally, it can be r e m a r k e d that the a n t i - a - t u b u l i n sequence (aS) antibodies yield a spread staining on Paramecium axonemal ~x-tubulin, extending all the way to the level of the ~-subunit, as the anti-acetylated tubulin antibodies do (fig 2A). In contrast, on Paramecium cytoplasmic and pig brain ~-tubulins, these antibodies labeled fine bands (figs 7B and 213). This suggests that the c~S antibodies recognize, in axonemal tubulin, different a-tubulin isoforms resulting from various axonemal-specific post-transl a t i o n a l m o d i f i c a t i o n s that w o u l d be c a u s i n g slight differences in eleetrophoretic migrations (see for example, the shifts of migration of acetylated pig brain tubulin, in fig 11). The lack (or weakness) of reaction of the smear on Paramecium axonemes with other anti-sequence antibodies
such as D M I A and D M I B could be attributed to their insufficient affinity for the minor isoforms which are detected by Coomassie blue only on highly loaded gels. In conclusion, although some uncertainty remains as to the precise amino-acids involved, the location of the epitopes recognized by our anti-sequence antibodies has been defined within a relatively short peptide domain. The A X O 45 ° anti-~-tubulin antibody: a presumptive antisequence probe
In the absence of ~-tubulin fusion proteins, the discrimination between anti-fl sequence and anti-fl modification antibodies could only be based on immunocytochemical arguments. T h e r e f o r e the d e s i g n a t i o n of A X O 45 ° as an anti-sequence antibody was based on the similarity between its labeling pattern and that of reference anti-sequence antibodies, such as DM1A and DM1B. The epitope has been localized on a 21-kDa peptide starting at residue 257, also recognized by DM1B, ie situated in the C-terminal domain of the ~-tubulin molecule (fig 3). Since the reactivity of AXO 45 °, as of DM1B, is
116
AM Callen et al
much weaker on this peptide than on the whole molecule, it can be inferred that both antibodies recognize conformational determinants in addition to a sequence one. This agrees with the respective observations of Breitling and Littie [11] and of De la Vina et al [17] on the decrease of the reactivity of DM1B on fl-tubulin peptides and on a synthetic peptide 13 (412--431) in comparison with that on whole tubnlin. The case of anti-sequence antibodies that only react very weakly on fusion proteins
Two antibodies TAP 956 ° and TAP 926G, both directed against tx and fl-tubulins, yield immunocytochemical decoration patterns on Paramecium and metazoan cells very similar to those of the bona fide anti-sequence antibodies just discussed; but, in contrast to the latter, they react only with very high amounts of Stylonychia fusion protein (21A. A,d5). This weak response appears to be significant for several reasons. First, increasing the amounts of fusion protein loaded on a gel in the same proportions never yielded positive results with antibodies known with certainty to be directed against post-translational modifications (see below). Second, the central location in Paramecium ct- and fl-tubulins of the smallest peptides reactive with TAP 956 o and TAP 926G, at positions 159-281, and 188-310, respectively (fig 3) in a region where no post-translational modification has yet been found, suggests that the epitopes recognized by these antibodies correspond to sequence determinants. Third, at least some parts of this region are immunogenic, as evidenced by: i) the isolation of the antibodies C 98 and C 113 from synthetic peptides t~ (214-226) and fl (241-256) chosen from theoretical antigenlcity predictions [17]; and ii) the obtention, using microtubules or cleaved tubulin as immunogen, of antibodies directed to the central domains of both tx- and fl-tubulins [30, 74]. Because the two antibodies react both on Paramecium ani] mammalian ix- and fl-tubulins, we searched aligned sequences of both tx- and fl-tubulins in the 160o310 domain for strongly similar amino-acid stretches that might constitute the epitope (fig 14B). Three such stretches were found: tz(180-186) and fl(178-184), a(201-213) and fl(199-211), and tx(258-267) and fl(256-265). Comparison with the stretches against which De la Vina et al [17] generated their anti-synthetic peptide antibodies is quite interesting. The antibodies they obtained, directed towards the peptides a(214-226) and/3(241-256), contrary to ours; turned out to be subunit-specific. A third antibody, C 140, directed to fl(153-165), was not reactive with the 6-kDa Paramecium fl-peptide (fig 3). This implies that these stretches should be excluded as potential epitopes for our subunit cross-reacting antibodies, thus narrowing down the possible localization of the epitopes and nicely fits with the broad locations we postulated. Furthermore, the fact that the antibodies are weakly positive on a 25-fold increased loading of a Stylonychia fusion peptide starting at position 214, suggests that of the three possible amino-acid domains, the region ct(258-267), ]3(256--265) could, at least partly, constitute the epitope. In that framework, the very weak reactivity of TAP 956 ° and TAP 926G on fusion proteins containing the presumptive epitope could be interpreted as being due either to direct steric hindrance or to an indirect eonformational effect on the epitope caused by the associated large fragment of fl-galactosidase. In summary, the strong or weakly positive reactions over fusion proteins, always corroborated by other data, specially immunocytochemical observations, allows to unambigu-
ously identify antibodies directed to amino-acid sequences, in a broad sense. This class being thus delineated, four other specially interesting types of antibodies which all display several properties different from the ones just described were identified. Antibodies directed against four distinct types of post-translational modifications
These antibodies were totally unreactive over fusion proteins, even if huge amounts are challenged (this is evidenced in fig 4C, AXO 58), in clear contrast with the antibodies classified as 'anti-sequence': they could be directed against either conformational or post-translational epitopes. However, they attracted our attention because of some similarities in the pattern of decoration of Paramecium cells they yielded as compared to those of reference antibodies directed against post-translational modifications. They can be further subdivided into two classes: the first is directed against an epitope the nature of which is identified, acetylation, and the second is directed to still unidentified epitopes. Antibodies to acetylated oc-tubulin. We have very clearly shown that three antibodies TEU 318 °, TEU 348 ° and TAP 931E behave exactly as 6-11B-1 antibody whose epitope is generated by acetylation of lysine 40 on t~-tubulin [44, 45]: they are totally negative on fusion proteins (fig 4B), decorate stable microtubules both in Paramecium and in metazoan cells, fail to label the microtubules of PtK2 cells, and weakly recognize Paramecium cytoplasmic tubulin and that of pig brain. All three, as 6-11B-1, became strongly reactive over chemically acetylated pig brain tubulin. In the conditions used, the main acetylated amino-acids are reported to be lysine and tyrosine residues but the latter were further deacetylated. Through peptide map immunoblotting, the epitope(s) recognized by the three antibodies was (were) located in the 1-100 region of the N-terminal domain of Paramecium ottubulin (no labeling of fl-tubulin peptides was ever observed). This region contains only three lysine residues, at positions 40, 60 and 96. Through direct sequencing, LeDizet and Piperno [44] have established that acetylation occurred on lysine 40 in Chlamydomonas tx-tubulin; an identical localization was found by Edd6 et al [23] on pig brain tubulin. Moreover, given the identity of Paramecium, Chlamydomonas and mammalian tx-tubulin sequences surrounding lysine 40, it is very tempting to conclude that our three antibodies are directed to an acetylation of lysine 40 occurring in Paramecium tubulin. However, we cannot formally exclude that lysine 60 or 96 could be involved. Antibodies to post-translational modifications other than acetylation In contrast to all the anti-sequence and anti-acetylated tubulin antibodies which all clearly decorated only the tip of Paramecium axonemes under our fixation conditions, the three antibodies, TAP 952 °, AXO 58, and AXO 49 ° yield a differential decoration of axonemes (fig 8). The proximal decoration of axonemes by AXO 58 is reminiscent of that provided by the monoclonal antibody GT 335 [10], directed against tubulin polyglutamylation [75]. The strong decoration of axonemes by AXO 49 ° is very similar to that of a previously characterized polyclonal serum raised against Paramecium axonemal tubulin, most probably directed
Tubulin diversity in Paramecium against a post-translational modification [2], while the labeling pattern of axonemes by TAP 952 ° along their whole length is intermediate between the two other ones. In addition, the three antibodies recognize only a specific subset of the other microtubular networks: TAP 952 ° has a very broad reactivity, AXO 58 decorates a more restricted subpopulation of microtubules and AXO 49 ° the narrowest and the most stable one. Finally, and importantly, a lag time is observed between assembly of new microtubular structures after division and their acquisition of reactivity to the AXO 49 ° antibody, as was observed with the polyclonal anti-Paramecium axonemal tubulin serum [2], providing a decisive argument in favour of the idea that it is directed to a post-translationally added epitope. Thus, in order to account for the differential labeling patterns of axonemes, it may be hypothesised that the modification recognized by AXO 49 ° leads to an apparent inacessibility of DM1A, DM1B, o~S, flS and Ac antibodies to their epitopes along the axoneme of Paramecium (except at the tip), ie precisely where the reactivity of AXO 49 ° is found. We have at present localized the presumed post-translational epitopes of three antibodies in a large C-terminal domain of both ¢x- and ~tubulins. Interestingly, it is in this region, at the C-terminal end, that all the known post-translational modifications of tubulin have been discovered (except for acetylation). Such is the case £or: i) detyrosination of metazoan ¢x-tubulin, but no C-terminal tyrosine is encoded by the known sequences of ciliates; ii) phosphorylation of ~tubulin; and iii) polyglutamylation, found in mammalian c~- and ~tubulins respectively at positions 445 and 435 (or 438). Only this modification has been shown to occur on both ¢x- and ~-tubulins, at their C-terminal ends. Some modifications revealed by our antibodies could be of the same nature as these. For example, AXO 58 displays many of the characteristics of the anti-glutamylation antibody GT 335. It is therefore tempting to hypothesize that AXO 58 is directed against tubulin glutamylation, the differences in reactivity pattern with respect to GT 335 being due to the fact that the two antibodies react preferentially with different forms of glutamylation. The same comment can be made for TAP 952 °. In contrast, for AXO 49 ° , the specificity corresponds very closely to that of the anti-Paramecium axonemal tubulin polyclonal serum and all the evidence, partly provided here and detailed in a forthcoming paper (Levilliers et al, in preparation), points to a new type of post-translational modification specially occurring on axonemes and also on a few other very stable microtubules and displaying a broad phylogenetic distribution. Two comments can be made on the differential expression of antigenic sites of tubulin. First, the anti-sequence antibodies we have recovered are directed to epitopes located either in a 30-kDa N-terminal/central ¢x peptide or in a 21-kDa C-terminal fl peptide, which precisely correspond to the most stable domains of tubulin, as reported by Mandelkow et al [49]; these would be long-lived within the immunized animal relative to the other ones, and hence more prone to induce an immune response. Second, the antigenic determinants corresponding to post-translational m o d i f i c a t i o n s and t h o s e c o r r e s p o n d i n g to p r i m a r y sequences seem to be mutually exclusive. Indeed, as conterns the C-terminal domain of axonemal tubulin, which is exposed and probably immunogenic (this being confirmed by the existence of the DM1A and DM1B antibodies and other anti-sequence ones (for review see [4]), we have obtained only one anti-sequence fl and no anti-sequence but three anti (¢x + fl)-tubulin antibodies p r e s u m a b l y
117
directed against distinct post-translational modifications. If these modifications are bulky, they may be immunodominant and could mask the potential sequence determinants, all the more that axonemal tubulin may be highly modified. In conclusion, we have produced a number of anti-tubulln antibodies which are directed either against primary sequences or against post-translational epitopes and we have provided a number of congruent circumstantial evidence indicating that several of the latter antibodies belong to previously undescdbed specificities. While the panel is not exhaustive, it is already sufficiently large and the biochemical characterization of the epitopes is sufficiently advanced for these antibodies to be used as tools of defined specificity both for detailed immunocytochemical studies and for further biochemical characterization of the epitopes. Acknowledgments This work was supported by grants from the CNRS, the 'Universit~ Paris-Sud', the 'Action Inttgrte Franco-Espagnole n° 91047', and the 'Direccion General de Investigacion cientifica y t~cnica (DGICYT), project PB 91/0621'. This work could not have been carried out without the generous gifts of samples and antibodies provided by the many colleagues listed in Materials and methods, to whom we are grateful. We thank Herv6 Le Guyader for this contribution at the start of work, Herr6 Philippe for help with computing and for sharing aligned sequences, and Michel Laurent for his assistance in confocal microscopy and image processing. We would also like to thank No,lie Narradon for all the photographic work, and CEcile Couanon for her extensive help in editing the manuscript. References
1 Adoutte A, Claisse M, Maunoury R, Beisson J (1985) Tubulin evolution: Ciliate-specific epitopes are conserved in the ciliary tubulin of metazoa. J Mol Evo122, 220-229 2 Adoutte A, Delgado P, Fleury A, Levilliers N, Lain6 MC, Marry MC, Boisvieux-UMch E, Sandoz D (1991) Microtubule diversity in ciliated cells: evidence for its generation by post-translational modification in the axonemes of Paramecium and quail oviduct cells. Biol Cell 71,227-245 3 Alexander JE, Hunt DF, Lee MK, Shabanowitz J, Michel H, Berlin SC, Macdonald TL, Sundberg RJ, Rebhun LI, Frankfurter A (1991) Characterization of posttranslational modifications in neuron-specific class HI ~-tubulin by mass spectrometry. Proc Natl Acad Sci USA 88, 4685-4689 4 Andreu JM, de Pereda JM (1993) Site-directed antibodies to tubulin. Cell Motil Cytoskeleton 26, 1-6 5 Axevalo MA, Nieto JM, Andreu D, Andreu JM (1990) Tubulin assembly probed with antibodies to synthetic peptides. J Mol Bio1214, 105-120 6 Barahona I, Soares H, Cyrne L, Penque D, Denoulet P, Rodrigues-Pousada C (1988) Sequence of one ~- and two ~-tubulin genes of Tetrahymena pyriformis. Structural and functional relationships with other tubulin genes. J Mol Biol 202, 365-382 7 Barra HS, Arce CA, Rodriguez JA, Caputto R (1974) Some common properties of the protein that incorporate tyrosine as a single unit and the mierotubule proteins. Biochem Biophys Res Commun 60, 1384-1390 8 Best D, Wart PJ, Gull K (1981) Influence of the composition of commercial sodium dodecyl sulfate preparations on the separation of ~- and ~tubulin during polyacrylamide gel electrophoresis. Anal Biochem 114, 281-284 9 Blose SH, Melzer DI, Feramisco JR (1984) 10-rim filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Bio198, 847-858
118
AM Callen et al
10 Br6 MH, de Ntchaud B, Wolff A, Fleury A (1994) Glutamylated tubulin probed in eiliates with the monoclonal antibody GT 335. Cell Motil Cytoskeleton 27, 337-349 11 Breitling F, Little M (1986) Carboxy-terminal regions on the surface of tubulin and microtubules. Epitope locations of YOL 1/34, DMIA and DMIB. J M o l Biol 189, 367-370 12 Cleveland DW (1987) The multitubulin hypothesis revisited: What have we learned? J Cell Biol 104, 381-383 13 Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel eleetrophoresis. J Biol Chem 252, 1102-1106 14 Cohen J, Adoutte A, Grandchamp S, Houdebine LM, Beisson J (1982) Immunocytochemical study of microtubular structures throughout the cell cycle of Paramecium. Biol Cell 44, 35-44 15 Cohen J, Beisson J (1988) The cytoskeleton. In: 'Paramecium' (Gtrtz HD, ed) Springer-Verlag, Berlin - Heidelberg, 363-392 16 De Jong WW, Zweers A, Cohen LH (1978) Influence of single amino acid substitutions on electrophoretic mobility of sodium dodec.yl sulfate-protein complexes. B i o c h e m Biophys Res Commun 82, 532-539 17 De la Vina S, Andreu D, Medrano FJ, Nieto JM, Andreu JM (1988) T~bulin structure probed with antibodies to synthetic peptides. Mapping of three major types of limited proteolysis fragments. Biochemistry 27, 5352-5365 18 Draber P, Draberova E, Linhartova I, Viklicky V (1989) Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domainspecific monoclonal antibodies. J Cell Sci 92, 519-528 19 Drapeau GR (1976) Protease from Staphyloccus aureus. Methods Enzymo145, 469--475 20 Dupuis P (1992) ~Structure, Organisation et Expression des GEnes de Tubuline chez la Paramtcie~. Doctoral Thesis, Universit6 Paris XI, Orsay 21 Dupuis P (1992) The beta-tubulin genes of Paramecium are interrupted by two 27 bp introns. EMBO J I1, 3713-3719 22 Edd6 B, Jeautet C, Gros F (1981) One ]~-tubulin subunit accumulates during neurite outgrowth in mouse neuroblastoma ceils. Biochem Biophys Res Commun 103, 1035-1043 23 Edd6 B, Rossier J, Le Caer JP, Berwald-Netter Y, Koulakoff A, Gros F, Denoulet P (1991) A combination of posttranslational modifications is responsible for the production of neuronal a-tubulin heterogeneity. J Cell Biochem 46, 134-142 24 Edd6 B, Rossier J, Le Caer JP, Desbruy~res E, Gros F, Denoulet P (1990) Posttranslational glutamylation of atubulin. Science 247, 83-85 25 Eipper BA (1974) Properties of rat brain tubulin. J Biol Chem 249, 1407-1416 26 Fleury A (1991) Dynamics of the cytoskeleton during morphogenesis in the Ciliate Euplotes. I. Basal bodies related microtubular system. Eur J Protisto127, 99-114 27 Fling SP, Gregerson DS (1986) Peptide and protein molecular weight determination by electrophoresis using a high-molarity Tris buffer system without urea. Anal Biochem 155, 83-88 28 Forgue ST, Dalai JL (1979) Rat brain tubulin: tubulin heterogeneity and phosphorylation. J Neurochem 32, 1015-1025 29 Gaertig J, Thatcher TH, McGrath KE, Callahan RC, Gorovsky MA (1993) Perspectives on tubulin isotype function and evolution based on the observation that Tetrahymena thermophila microtubules contain a single ¢x-and ~tubulin. Cell Motil Cytoskeleton 25, 243-253 30 Gallo JM, Anderton BH (1983) A subpopulation of trypanosome mierotubules recognized by a monoclonal antibody to tubuUn. EMBO J 2, 479--483 31 Gard DL, Kirsehner MW (1985) A polymer-dependent increase in phosphorylation of ~tubulin accompanies differentiation of a mouse neuroblastoma cell line. J Cell Biol 100, 764-774 32 Geuens G, Hill AM, Levillier~ N, Adoutte A, DeBrabander M (1989) Mierotubule dynamics investigated by microinjoction of Paramecium axonemal tubulin: Lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase. J Cell Biol 108, 939-953
33 Greer K, Rosenbaum JL (1989) Post-translational modifications of tubulin. In: Cell movement. Vol 2: Kinesin, Dynein and Microtubule Dynamics (Warner FD, Macintosh JR, eds) Alan R Liss, New York, 47--66 34 Grimm M, Breitling F, Little M (1987) Location of the epitope for the cz-tubulin monoclonal antibody TU-01. Biochim Biophys Acta 91, 4 83-88 35 Gull K, Hussey PJ, Sasse R, Schneider A, Seebeck T, Sherwin T (1986) Tubulin isotypes: Generation of diversity in cells and microtubular organelles. J Cell Sci (Suppl) 5, 243-255 36 Helftenbein E, MUller E (1988) Both a-tubulin genes are transcriptionally active in Stylonychia lemnae. Curr Genet 13,425--432 37 Hoyle HD, Raft EC (1990) Two Drosophila beta tubulin isoforms are not functionally equivalent. J Cell Biol 111, 1009-1026 38 Huang J, Matthews HR (1990) Application of sodium dodecyl sulfate-gel electrophoresis to low molecular weight polypeptides. Anal Biochem 188, 114--117 39 Jeanmaire-Wolf R, Cltrot JC, Nahon P, Iftode F, Fleury A, Adoutte A (1993) Isolation and characterization of monoclohal antibodies to cytoskeletal and membrane proteins of the Paramecium cortex. Eur J Protisto129, 311-333 40 Joshi HC, Cleveland DW (1990) Diversity among tubulin subunits: toward what functional end? Cell Motil Cytoskeleton 16, 159-163 41 Kopczak SD, Haas NA, Hussey PJ, Silflow CD, Snustad DP (1992) The small genome of Arabidopsis contains at least six expressed a-tubulin genes. Plant Cell 4, 539-547 42 Kyhse-Andersen J (1984) Eloctroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitroceUulose. J Biochem Biophys Methods 10, 203-209 43 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680--685 44 LeDizet M, Piperno G (1987) Identification of an acetylation site of Chlamydomonas /x-tubulin. Proc Natl Acad Sci USA 84, 5720-5724 45 LeDizet M, Piperno G (1991) Detection of acetylated a-tubulin by specific antibodies. Methods Enzymol 196, 264-274 46 L'Hemault SW, Rosenbaum JL (1983) Chlamydomonas ottubulin is posttranslationally modified in the flagella during flagellar assembly. J Cell Bio197, 258--263 47 L'Hemault SW, Rosenbaum JL (1985) Chlamydomonas ottubulin is posttranslationally modified by acetylation on the e-amino group of a lysine. Biochemistry 24, 473--478 48 Luduena R.F, Zimmermann HP, Little M (1988) Identification of the phosphorylated ~tubulin isotype in differentiated neuroblastoma cells. FEBS Lett 230, 142-146 49 Mandelkow EM, Herrmann M, Rtihl U (1985) Tubulin domains probed by limited proteolysis and subunit-specific antibodies. J Mol Biol 185, 311-327 50 Merril CR, Goldman D, Sedman SA, Ebert MH (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437-1438 51 Merril CR, Goldman D, Van Keuren ML (1983) Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymo196, 230-239 52 Paturle-Lafanech~re L, Edd6 B, Denoulet P, Van Dorsselaer A, Mazarguil H, Le Caer JP, Wehland J, Job D (1991) Characterization of a major brain tubulin variant which cannot be tyrosinated. Biochemistry 30, 10523-10528 53 Pipet'no G, Fuller MT (1985) Mouoelonal antibodies specific for an acetylated form of ¢x-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol 101, 2085-2094 54 Piperno G, LeDizet M, Chang X (1987) Microtubules containing acetylated ¢x-tubulin in mammalian cells in culture. J Cell Biol 104, 289-302 55 Raft EC (1984) Genetics of mierotubule systems. J Cell Biol 99, 1-10
Tubulin diversity in Paramecium 56 Raybin D, Flavin M (1975) An enzyme tyrosilating alphatubulin and its role in microtubule assembly. Biochem Biophys Res Commun 65, 1088-1095 57 Redeker V, Melki R, Prom6 D, Le Caer JP, Rossier J (1992) Structure of tubulin C-terminal domain obtained by subtilisin treatment. The major t~ and fl tubulin isotypes from pig brain are glutamylated. FEBS Lett 313, 185-192 58 Riordan JF, Vallee BL (1967) Acetylation. Methods Enzymol I 1,565-570 59 Riordan JF, Vallee BL (1967) O-Acetyltyrosine. Methods Enzymol 11,570-576 60 Rtidiger M, Plessman U, Kl6ppel KD, Wehland J, Weber K (1992) Class 1I tubulin, the major brain fl tubulin isotype is polyglutamylated on glutamic acid residue 435. FEBS Lett 308, 101-105 61 Sasse R, Gull K (1988) Tubulin post-translational modifications and the construction of microtubular organelles in Trypanosoma brucei. J Cell Sci 90, 577-589 62 Schliwa M, van Blerkom J (1981) Structural interaction of cytoskeletal components. J Cell Bio190, 222-235 63 Serrano L, Wandosell F, Avila J (1986) Location of the regions recognized by five commercial antibodies on the tubulin molecule. Anal Biochem 159, 253-259 64 Silflow CD, Oppenheimer DG, Kopczak SD, Ploense SE, Ludwig SR, Haas N, Snustad DP (1987) Structure and differential expression during development. Dev Genet 8, 435--460 65 Steinbrtick G (1990) Recent advances in the study of ciliate genes. Eur J Protisto126, 2-14 66 Sullivan KF (1988) Structure and utilization of tubulin isotypes. Annu Rev Cell Biol 4, 687-716
119
67 Suprenant KA, Hays E, l..eCluyse E, Dentler WL (1985) Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila. Proc Natl Acad Sci USA 82, 6908---6912 68 Swank RT, Munkres KD (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem 39, 462--477 69 Thompson WC (1982) The cyclic tyrosination/detyrosination of alpha tubulin. Methods Cell Bio124, 235-255 70 Torres A, Delgado P (1989) Effects of cold and nocodazole treatments on the microtubular systems of Paramecium in interphase. J Protozoo136, 113-119 71 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76, 4350--4354 72 Tung JS, Knight CA (1972) Relative importance of some factors affecting the electrophoretic migration of proteins in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 48, 153-163 73 Viklicky V, Draber P, Hasek J, Bartek J (1982) Production and characterization of a monoclonal antitubulin antibody. Cell Biol Int Rep 6, 725-731 74 Wolff A, de N6chaud B, Chillet D, Gros F (1988) The aminoterminal domain of alpha-tubulin probed by monoclonal antibodies. Recognization of a rarely exposed epitope by the monoclonal antibody 111 B52 C2. Biol Cell 63, 319-326 75 Wolff A, de N6chaud B, Chillet D, Mazarguil H, Desbruy~res E, Audebert S, Edd6 B, Gros F, Denoulet P (1992) Distribution of glutamylated tx and fl-tubulin in mouse tissues using a specific monoclonal antibody, GT 335. Eur J Cell Bio159, 425--432