- Email: [email protected]
d1 neuroblastoma cells
Developmental Brain Research, 51 (1990) 195-204 Elsevier
195
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Post-translational modification of a-tubulin by acetylation and detyrosination in NB2a/dl neuroblastoma cells Thomas B. Shea 1'2, Mary Lou Beermann I and Ralph A. Nixon t'3 1Ralph Lowell Laboratories, Mailman Research Center, McLean Hospital, Belmont, MA 02178 (U.S.A.), 2Departments of Biological Chemistry and Molecular Pharmacology and 3Program in Neuroscience, Harvard Medical School, Boston, MA 02115 (U.S.A.)
(Accepted 1 August 1989) Key words: Microtubule; a-Tubulin; Neuronal cell; Cytoskeleton; Acetylation; Detyrosination; Neuronal differentiation; Neurite maturation
Western blot analyses of total assembled microtubule fractions from NB2aJdl neuroblastoma cells demonstrated that these cells are capable of post-translationally modifying a-tubulin by acetylation and detyrosination. Immunocytochemical analyses of NB2a/dl cells differentiated with dbcAMP which had been processed under microtubule-stabilizing conditions demonstrated that all forms of a-tubulin were present throughout perikarya and neurites. By contrast, extraction of cells with Triton X-100 revealed a regional concentration of acetylated and detyrosinated a-tubulin subunits within axonal neurites, detectable in some cells after 3 days of differentiation and in nearly all cells after 7 days. Resistance of neurites to retraction following colchicine-treatment developed at a similar rate; furthermore, colchicine-resistant neurites contained intense acetylated a-tubulin immunoreactivity. We conclude that NB2a/dl cells are capable of acetylating and detyrosinating a-tubulin subunits and that selective post-translational modification of a-tubulin subunits may be related to neuritic maturation. INTRODUCTION Microtubules are thought to mediate a number of diverse cellular functions including locomotion, division, organelle and vesicular translocation, establishment and maintenance of polarity and morphology, and outgrowth and maintenance of neurites and non-neuronal processes (for review, see ref. 29). This functional diversity is achieved in part by heterogeneity in the structure of tubulin subunits, which in turn results from the existence of multiple tubulin genes 1°, and from the modification of tubulin subunits after synthesis. In addition to mediating the dynamic functions typical of most cells (above), neuronal microtubules also mediate axonal outgrowth and contribute to neuritic maintenance by interacting with the other constituents of the neuritic cytoskeleton 26"44. These diverse functions are apparently achieved in part by selective expression of tubulin isotypes and stabilization of specific microtubule subpopulations. A transient increase in the expression of m R N A s for certain a-tubulin isotypes accompanies nervous system development; this enrichment of expression is confined to regions of active neurite outgrowth 44. A similar increase in expression of m R N A coding for selective a-tubulin isotypes accompanies nerve growth factor-induced differentiation of PC12 cells 44. Subpopulations of relatively stable microtubules are concentrated
within axons 8. Axonally transported tubulin has been shown to contain a relatively slow-moving population of Triton-, cold- and colchicine-resistant microtubules which differs in subunit composition from the faster-transported tubulin p o o l 15'39'4°. Transport studies demonstrate that the slower-moving, stabilized microtubules co-migrate with neurofilaments; furthermore, when neurofilament transport was slowed by treatment with fl,fl'-iminodipropionitrile, transport of these stable microtubules was retarded to a similar extent, suggesting the presence of a physical association between neurofilaments and the stabilized microtubules 39. Several other studies have also documented the presence of cold-stable microtubule populations in n e u r o n s 4'29'42 and a regional concentration of cold-stable microtubules within axons 8'12. Cold-stable microtubules from brain contain a relatively higher concentration of two a-tubulin isotypes as compared to the overall microtubule population 22,39. Subcellular differentiation of populations of neuronal microtubules is also achieved in part by the selective post-translational modification of a-tubulin subunits by acetylation 7'31 and detyrosination 2"3'27'32. Immunocytochemical examinations of rat cerebral cortex, cerebellum, corpus callosum and brainstem demonstrated that acetyiated a-tubulin is present in neurons and is enriched in axons 9. Cultured sympathetic and granule cell neurons contain acetylated a-tubulin 6"9. The induction of acety-
Correspondence: T.B. Shea, Ralph Lowell Laboratories, McLean Hospital, Belmont, MA 02178, U.S.A.
196 l a t i o n of a - t u b u l i n has also b e e n d e m o n s t r a t e d in PC12 cells after l o n g - t e r m t r e a t m e n t with n e r v e growth factor 6. R e t i n a l m i c r o t u b u l e s c o n t a i n i n g acetylated (~-tubulin are more
resistant to d e p o l y m e r i z a t i o n by colchicine or
n o c a d a z o l e t h a n m i c r o t u b u l e s that did n o t c o n t a i n acetylated s u b u n i t s 33. T h e s e o b s e r v a t i o n s suggest that acety-
mM dibutyryl adenosine 3'.5'-cyclic monophosphate (dbcAMP; Sigma Chemical Co., St. Louis, MO) 24 h after plating. Medium was routinely changed every 3 days, with the inclusion of dbcAMP where appropriate. Untreated cells arc designated as NB2a(-): treated cells are designated as NB2a(+ 1), NB2a(+3) or NB2a(+7) to indicate the length in days of dbcAMP treatment. Cells were routinely examined and photographed by phase-contrast microscopy.
lation of a - t u b u l i n r e p r e s e n t s o n e m e c h a n i s m by which stability is c o n f e r r e d u p o n neuritic m i c r o t u b u l e s . D e t y r o s i n a t e d a - t u b u l i n s u b u n i t s are also c o n c e n t r a t e d w i t h i n n e u r i t e s 9'18. By contrast, a - t u b u l i n s u b u n i t s within m i c r o t u b u l e s in highly motile areas such as the a x o n a l g r o w t h c o n e are p r e d o m i n a n t l y t y r o s i n a t e d ~7. A l t h o u g h cold-
and
Triton-resistant microtubules
are
Colchicine treatment
Stability of neurites to colchicine was examined by incubation of cultures at 37 °C in the above medium containing 10-6 M colchicine for 2 h, after which they were examined by phase-contrast microscopy. In cases where neurites had not retracted, duplicate cultures were further incubated in the presence of colchicine for a total of 18 h.
typically
e n r i c h e d in d e t y r o s i n a t e d a - t u b u l i n s u b u n i t s , h o w e v e r , d e t y r o s i n a t i o n a l o n e is a p p a r e n t l y insufficient to confer stability to m i c r o t u b u l e s 23. Since m i c r o t u b u l e s c o n t a i n i n g significant a m o u n t s of d e t y r o s i n a t e d s u b u n i t s do n o t c o n t i n u e to e l o n g a t e 19'43, it has b e e n suggested that this s u b s e t of m i c r o t u b u l e s m a y p r e f e r e n t i a l l y be ' c a p p e d ' , a p h e n o m e n o n which confers e n h a n c e d stability a n d a l o n g e r half-life to m i c r o t u b u l e s by restricting t e r m i n a l s u b u n i t e x c h a n g e 17. A n t i b o d i e s specific for d e t y r o s i n a t e d a n d a c e t y l a t e d s u b u n i t s l a b e l e d a n identical m i c r o t u b u l e s u b p o p u l a t i o n in h u m a n r e t i n o b l a s t o m a cells, indicating that m i c r o t u b u l e s can c o n t a i n both d e t y r o s i n a t e d a n d a c e t y l a t e d s u b u n i t s ; the possibility r e m a i n s that s o m e a - t u b u l i n s u b u n i t s m a y have u n d e r g o n e both modific a t i o n s 35. M o u s e N B 2 a / d l n e u r o b l a s t o m a cells n o r m a l l y grow e x p o n e n t i a l l y a n d possess short, p u t a t i v e neurites. H o w ever, they can be i n d u c e d to e l a b o r a t e e i t h e r exclusively a x o n a l or d e n d r i t i c n e u r i t e s by t r e a t m e n t with dibutyryl cyclic A M P or r e t i n o i c acid, respectively 36. In the p r e s e n t study we d e m o n s t r a t e that m o u s e N B 2 a / d l n e u r o b l a s t o m a cells are c a p a b l e of acetylating a n d d e t y r o s i n a t i n g a - t u b u l i n s u b u n i t s . W e f u r t h e r d e m o n s t r a t e that the e l a b o r a t i o n of a x o n a l n e u r i t e s d u r i n g i n d u c e d differentiation of these cells is a c c o m p a n i e d by c o m p a r t m e n t a l ization of a c e t y l a t e d a n d d e t y r o s i n a t e d a - s u b u n i t s within cold- a n d T r i t o n - r e s i s t a n t a x o n a l m i c r o t u b u l e s ; this increase in p o s t - t r a n s l a t i o n a l l y m o d i f i e d s u b u n i t i m m u n o reactivity coincides with the a t t a i n m e n t of n e u r i t i c stability to colchicine t r e a t m e n t .
lmmunocytochemical analyses
Cultures were rinsed briefly in Tris-buffered saline (pH 7A; TBS) and fixed for 20 min with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) then rinsed 3 times with TBS. Cultures were either extracted with 1% Triton X-100 in TBS for 20 rain at room temperature or incubated under identical conditions without Triton, then incubated overnight at room temperature with mouse monoclonal antibody B5-1-2 (which reacts with all forms of a-tubulin), mouse monoclonal antibody 6-11B-1 (which reacts specifically with acetylated a-tubulin3°), rabbit polyclonal antiserum TYR (which reacts specifically with tyrosinated a-tubulin) and rabbit polyclonal antiserum GLU (which reacts specifically with detyrosinated atubulin19). The following day the cultures were rinsed 3 times in TBS (5 min each) and incubated with goat anti-rabbit or goat anti-mouse lgG conjugated with horseradish peroxidase, rinsed 3 times, and developed with diaminobenzidine in the presence of. H20 2, B5-1-2 and 6-11B-1 were generous gifts of Dr. G. Piperno, Rockefeller University, NY; TYR and GLU were generous gifts of Dr. Jeanette Chloe Bulinski, University of California, Los Angeles, CA. Isolation of total assembled microtubules and Triton-insoluble microtubules
To obtain the total assembled microtubule fraction38, cells were rinsed in TBS (30 °C), scraped from the plate with a rubber policeman in 0.1 M PIPES buffer (pH6.7) containing 1 mM MgC12, 2 mM EGTA, 0.1 mM EDTA. 2 M glycerol at 30°C and homogenized in a Dounce homogenizer at room temperature. The homogenate was centrifuged at 37.000 g for 15 rain at 30 °C. and the resulting pellet was rinsed in the same buffer and resuspended in 1% sodium dodecyl sulfate (SDS). Triton-insoluble cytoskeletons were prepared as previously described u'36. Cultures were rinsed with TBS (4 °C) then scraped from the plate with a rubber policeman in cold 1% Triton X-100 in 50 mM Tris-HCl (pH 6.8), 5 mM EDTA. 2 mM phenylmethylsulfonyl fluoride and 50/~g/ml leupeptin The cells were homogenized on ice in a Dounce homogenizer and centrifuged for 10 min at 13.000 g at 4 °C. The resulting pellet. defined as the Triton-insoluble cytoskeletal fraction, was rinsed in the same buffer without Triton. then resuspended in 1% SDS. lmmunoblot analysis
MATERIALS AND METHODS Cells and culture conditions
The NB2a/dl cell line36"37, a subclone of mouse NB2a neuroblastoma (originally derived from C1300 cells ~) was used in all experiments. Cells were plated in Dulbecco's Modified Eagle Medium containing 10% fetal calf serum and 25/~g/ml gentamycin (Gibco, Grand Island, NY) in either 10-cm plates or in multi-well chamber slides (Lab-Tek, Naperville, MD) in a humidified atmosphere of 95% air and 5% CO 2. To induce the outgrowth of axonal neurites36"37, the medium was replaced with medium containing 1
Samples were normalized with respect to total protein in the homogenate prior to fractionation, and were electrophoresed on 10% SDS gels according to Laemmli24. Nitrocellulose replicas were obtained as described41 in a Hoefer Transphor apparatus. Prestained molecular weight markers (myosin, 200 kDa; phosphorylase B, 97.4 kDa; bovine serum albumin. 68 kDa; ovalbumin. 43 kDa- achymotrypsinogen, 25.7 kDa: fl-lactoglobulin. 18.4 kDa; Bethesda Research Laboratories, Gaithersburg, MD) were included both for determination of molecular weights of immunolabeled proteins and to monitor the transfer of proteins to nitrocellulose. The proteins were visualized by sequential reaction of the replicas with 6-11B-I or B5-1-2 (1:20 dilution) or anti-GLU or anti-TYR (1:100 dilutionl
197
A B C
D
E
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antibody specific for tyrosinated a-tubulin subunits (Fig. 1D), and a-tubulin subunits which had been posttranslationally modified by detyrosination (Fig. 1C) and acetylation (Fig. 1E). In each case, a single polypeptide with an apparent molecular weight of approximately 52
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30Fig. 1. Characterization of anti-a-tubulin antibodies and antisera on NB2a/dl cytoskeletons, Electrophoretic analysis of total microtubule fractions of NB2a(-) cells. Lane A: representative Coomassie brilliant blue-stained gel. Lane B: nitrocellulose replica stained with monoclonal antibody B5-1-2 (specific for all a-tubulin subunits regardless of post-translationally modified state). Lane C: nitrocellulose replica stained with monoclonal antibody 6-11B-I (specific for acetylated subunits). Lane D: nitrocellulose replica stained with polyclonal antiserum TYR (specific for tyrosinated subunits). Lane E: nitrocellulose replica stained with polyclonal antiserum GLU (specific for detyrosinated subunits). The migration of molecular weight standards are noted on the left portion of the electrophoretegram. Note the immunostaining with all antibodies of a single polypeptide migrating at approximately 52 kDa.
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followed by goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (ICN Immunobiochemicals) followed by diaminobenzidine in the presence of H202 as previously described 16.
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RESULTS
Nitrocellulose replicas of the total assembled microtubule fractions from undifferentiated NB2a/dl cells were reacted with 4 different antibodies directed against a-tubulin subunits including an antibody which recognized all forms of a-tubulin subunits (Fig. 1B), an
Fig. 2. Distribution of assembled tyrosinated and detyrosinated a-tubulin subunits in NB2a/dl cells, a: phase-contrast image, b: unextracted culture stained with TYR. c: Triton-extracted culture stained with TYR. d: unextracted culture stained with GLU. e: Triton-extracted culture stained with GLU. Note that tyrosinated a-tubulin immunoreactivity is present throughout perikarya and neurites of cells before (b) and, at a reduced level, after (c) extraction. By contrast, although detyrosinated a-tubulin immunoreactivity is also present throughout perikarya and neurites of cells before (d) and after (e) extraction, only the perikaryal detyrosinated a-tubulin immunoreactivity is substantially reduced following extraction, while that of axonal neurites (arrows) is largely retained.
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198 kDa was observed, indicating that NB2a/dl cells can acetylate and detyrosinate a-tubulin subunits, and also contain a population of tyrosinated a-tubulin subunits. We next examined the intracellular distribution of a-tubulin subunits in differentiated NB2a/dl cells. Since neurons have subsets of microtubules which differ in stability, we performed immunocytochemical analyses on cells that had been processed either under conditions that stabilized all microtubules (30 °C, in the absence of Triton) or that extracted all but the subset of cold- and Triton-resistant microtubules (4 °C, in the presence of 1% Triton). Tyrosinated a-tubulin subunits were present throughout perikarya and neurites of cells harvested under microtubule-stabilizing conditions (Fig. 2a,b). Extraction of cells with Triton before immunocytochemical analyses reduced the overall level of staining; however, the extracted cells retained a similar relative distribution of tyrosinated subunit immunoreactivity (Fig. 2c). Detyrosinated subunits were also present throughout perikarya and neurites of cells processed under microtubulestabilizing conditions (Fig. 2d). However, immunoreactivity in perikarya was substantially reduced following extraction, while that of axonal neurites (arrows) underwent less reduction. Similarly, acetylated subunits were present throughout perikarya and neurites of unextracted cells (Fig. 3c); extraction of cells decreased only perikaryal immunoreactivity and did not substantially reduce acetylated a-tubulin immunoreactivity in neurites (Fig. 3d). Total a-tubulin immunoreactivity was relatively uniformly dispersed throughout perikarya and neurites in unextracted cells (Fig. 3a) and was reduced in extracted cells (Fig. 3b). The decrease in total a-tubulin immunoreactivity in neurites of extracted cells indicated that the high levels of detyrosinated and acetylated tubulin staining in neurites of similar cells does not simply reflect greater numbers of neuritic microtubules alone, but rather a selective compartmentalization of detyrosinated and acetylated subunits within cold- and Triton-resistant microtubules in neurites. Outgrowth of axonal neurites is rapid over the first 3 days following the addition of dbcAMP to NB2a/dl cells, after which neurites continue to elongate at a substantially slower rate 36'37 (see also Fig. 6). We therefore examined Triton-extracted NB2a/dl cells at several time points during differentiation to determine whether compartmentalization of acetylated and detyrosinated atubulin subunits within Triton-insoluble axonal microtubules accompanied the initial rapid phase of neurite outgrowth or was delayed. Perikaryal immunoreactivity against acetylated a-tubulin remained relatively constant throughout the first 7 days of differentiation (Fig. 4). The putative neurites of NB2a(-) cells (Fig. 4a) and the short neurites elaborated by NB2a(+I) cells (Fig. 4b) stained
at levels similar to their respective perikarya. By contrast, while a broad range of staining intensity was observed among individual neurites within each culture, some neurites of NB2a(+3) cells (Fig. 4e, small arrows) and most neurites of NB2a(+7) cells (Fig. 4g) exhibited more staining than their respective perikarya. No regional compartmentalization of total a-tubulin immu-
a
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Fig. 3. Distribution of total and acetylated a-tubulin subunits in NB2a/dl cells, a: unextracted culture stained with B5-1-2. b: Triton-extracted culture stained with B5-1-2. c: unextracted culture stained with 6-11B-1. d: Triton-extracted culture stained with 6-11B-1. Note that immunoreactivity towards total a-tubulin is present throughout perikarya and neurites of cells before (a) and, at a reduced level, after (b) extraction. Acetytated a-tubulin immunoreactivityis also present throughout perikarya and neurites of cells before (c) and after (d) extraction; however, while perikaryal immunoreactivityis reduced followingextraction, immunoreactivity within neurites (arrows) is only slightly reduced.
199
noreactivity occurred; perikarya and their respective neurites exhibited similar levels of staining at all times during differentiation (Fig. 4b,d,f,h).
Detyrosinated a-tubulin immunoreactivity remained relatively constant within perikarya throughout differentiation (Fig. 5). The putative neurites of NB2a(-) ceils
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Fig. 4. Regional concentration of acetylated a-tubulin subunits within Triton-resistant axonal microtubules during differentiation of NB2a/dl cells. Triton-extracted cultures after 0 (a,b), 1 (b,c,), 3 (e,f) or 7 days (g,h) in the presence of dbcAMP. Panels on the left (a,c,e,g) present cultures immunostained with 6-11B-1; panels on the right (b,d,f,h) present cultures stained with B5-1-2. Note that neurites (small arrows) of day 3 and day 7 cells (e.g) contained more acetylated a-tubulin immunoreactivity than perikarya (large arrows); by contrast, total a-tubulin immunoreactivity remained evenly dispersed throughout perikarya and neurites during differentiation (b,d,f,h).
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(Fig. 5a) and the short neurites elaborated by N B 2 a ( + I ) cells (Fig. 5c) stained at levels similar to their respective perikarya. By contrast, neurites (small arrows) of N B 2 a ( + 3 ) and (+7) cells were more stained than perikarya (Fig. 5e,g, large arrows). Perikarya exhibited similar levels of tyrosinated a-tubulin immunoreactivity as their respective neurites at all times during differentiation (Fig. 5b,d,f,h).
To determine the stage when neurite stability develops in NB2a/dl cells, we examined whether or not NB2a/dl neurites developed resistance to retraction following treatment with the microtubule-depolymerizing drug colchicine. The relatively short neurites present by the first day of differentiation (Fig. 6b) were rapidly retracted by treatment with colchicine (Fig. 6c). By the 3rd day of differentiation (Fig. 6d), however, NB2a/dl
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Fig. 5. Regional concentration of detyrosinated a-tubulin subunits within Triton-resistant axonal microtubules during differentiation of NB2a/dl cells. Triton-extracted cultures after 0 (a,b), 1 (b,c), 3 (e,f) or 7 days (g,h) in the presence of dbcAMP. Panels on the left (a,c,e,g) present cultures immunostained with GLU; panels on the right (b,d,f,h) present cultures stained with TYR. Note that neurites (small arrows) of day 3 and day 7 cells (e,g) contained more detyrosinated a-tubulin immunoreactivity than perikarya (large arrows); by contrast, tyrosinated a-tubulin immunoreactivity remained evenly dispersed throughout perikarya and neurites during differentiation (b,d,f,h).
201
Fig. 6. Development of colchicine-resistance of NB2a/dl axonal neurites during differentiation. NB2a/dl cells cultures for 0 (a), 1 (b,c,), 3 (d,e) or 7 days (f,g) in the presence of dbcAMP, after which time alternate cultures were treated with colchicine (c,e,g). The short neurites of NB2a(+ 1) cells were completely retracted by treatment with colchicine (c). Neurites of day 3 cells were partially resistant to colchicine; while some neurites were completely retracted, some neurites were only partially retracted (arrows), and others seemed relatively unaffected (e). Neurites of day 7 cells were resistant to colchicine treatment (g).
neurites (Fig. 6d) had developed a degree of resistance to colchicine (Fig. 6e). Although some neurites of colchicine-treated cells had completely retracted, many had only partially retracted (Fig. 6e, arrows), and still other neurites were indistinguishable by phase-contrast microscopy from those in non-colchicine-treated N B 2 a ( + 3 ) cultures. By the 7th day of differentiation (Fig. 60, virtually all neurites were apparently unaffected by colchicine treatment as ascertained by phase-contrast microscopy (Fig. 6g). Continued exposure to colchicine for 18 h resulted in the retraction of virtually all neurites in N B 2 a ( + 3 ) cultures; however, a small percentage of
neurites in N B 2 a ( + 7 ) cultures were resistant to this extended colchicine treatment (Fig. 7b). To investigate possible mechanisms for the development of colchicine-resistance, we analyzed Triton-extracted cells for the continued presence of acetylated a-tubulin in neurites surviving colchicine-treatment. N B 2 a ( + 3 ) and (+7) cultures were treated with colchicine for 2 and 18 h, respectively, extracted with Triton, and immunostained with monoclonal antibody 6-11B-1. The Triton-insoluble cytoskeleton of colchicine-resistant neurites of N B 2 a ( + 3 ) cells (Fig. 7a) and N B 2 a ( + 7 ) cells (Fig. 7b) contained intense acetylated a-tubulin immu-
202
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Fig. 7. Colchicine-resistant neurites contain acetylated a-tubulin. Triton-extracted NB2a(+3) and (+7) cells immunostained with 6-11B-1 following treatment with colchicine for 2 (a) and 18 h (b). Note that colchicine-resistantneurites (arrows) contain more acetylated a-tubulin immunoreactivity than their respective perikarya. noreactivity, indicating that colchicine-resistant neurites contain significant amounts of acetylated a-tubulin. DISCUSSION In the present study we demonstrate that NB2a/dl cells are capable of post-translationally modifiying atubulin by acetylation and detyrosination. Subunits which had been acetylated and detyrosinated were assembled within both Triton-labile and Triton-resistant microtubules; however, these two subsets of microtubules displayed a different subcellular distribution. Triton-labile microtubules containing acetylated and detyrosinated subunits were relatively evenly distributed throughout perikarya and neurites. By contrast, Triton-resistant microtubules containing acetylated and detyrosinated subunits were concentrated within neurites of differentiated cells. This regional concentration of acetylated and detyrosinated a-tubulin subunits within axonal neurites exhibited a delayed appearance during NB2a/dl differentiation. This differential distribution was not simply a reflection of increased numbers of Triton-resistant microtubules in neurites, since extraction decreased the level of immunostaining in both perikarya and neurites by a monoclonal antibody (B5-1-2) that recognizes all forms of a-tubulin. Tyrosinated a-tubulin subunits displayed a different immunocytochemical profile in extracted cells than did subunits that had been post-translationally modified by detyrosination. Following extraction, tyrosinated immu-
noreactivity declined in both perikarya and neurites. This effect indicates that, unlike detyrosinated subunits, tyrosinated subunits are not selectively concentrated within Triton-resistant microtubules of axonal neurites, q~hese results therefore suggest that the majority of ~-tubulin subunits in Triton-resistant axonal microtubules have been detyrosinated. Similarly, the overall depletion of total a-tubulin immunoreactivity following Triton-extraction indicates that the majority of a-tubulin subunits in NB2a/dl cells exists in a Triton-soluble form, either as unassembled subunits or assembled into Triton-labile microtubules. These data are in agreement with studies in other neuronal systems, where detyrosinated a-tubulin subunits were observed to be concentrated within neurites 9'1s, while tyrosinated subunits were localized within more motile cellular areas such as growth cones tv. Regional compartmentalization of acetylated a-tubulin subunits was not detected in previous studies of cultured granule cells9, sympathetic neurons or PC12 cells°; perikarya and neurites of these cells displayed approximately equivalent levels of immunoreactivity within perikarya and neurites. It should be noted, however, that these cultures were not Triton-extracted prior to immunocytochemical analyses. Similarly, unextracted NB2a/dl cells did not exhibit regional concentrations of acetylated and detyrosinated subunits; rather, in cells harvested under microtubule-stabilizing conditions, immunoreactivity towards a-tubulin subunits which had been acetylated and detyrosinated was present throughout perikarya and neurites. Triton-extraction of NB2a/dl cells, however, revealed a selective population of stabilized axonal microtubules which were enriched in acetylated and detyrosinated subunits. It is possible that, where similar extraction procedures performed on primary neuronal cultures or PC12 cells, regional concentration of post-translationally modified a-tubulin would be observed within neurites. Neurites of NB2a(+I) cells were rapidly retracted following exposure to colchicine, while neurites of NB2a(+3) and (+7) cells developed a progressive resistance to retraction following colchicine-treatment. Since regional concentration of acetylated a-tubulin subunits within neurites was not detected until 3 days after the addition of dbcAMP, it remains a possibility that the relatively high concentration of acetylated a-tubulin in microtubules of NB2a(+3) and NB2a(+7) neurites contributes to the development of neurite stability. Previous studies, however, have indicated that acetylation and detyrosination of a-tubulin are apparently not prerequisites for microtubule stability, and that stabilization of microtubules is not necessarily preceded by subunit acetylation or detyrosination. For example, while numerous chick embryo fibroblast microtubules contain dety-
203 r o s i n a t e d a - t u b u l i n subunits, these cells possess few stable microtubules. Conversely, kidney cells of P o t o r o u s tridactylis do not contain acetylated subunits, yet possess n u m e r o u s stable microtubules. Accordingly, m o d u l a t i o n of subunit t u r n o v e r has b e e n p r o p o s e d as a m a j o r factor that underlies the d e v e l o p m e n t of stability in select microtubules35; microtubules in which subunit exchange occurs at a slower rate could therefore accumulate a g r e a t e r p r o p o r t i o n of post-translationally modified subunits, and could exhibit greater resistance to d e p o l y m e rizing t r e a t m e n t s such as cold or colchicine. In addition, differential binding of M A P s and/or interactions with o t h e r cytoskeletal constituents such as neurofilaments m a y influence microtubule stability. T h e gradual d e v e l o p m e n t of colchicine resistance in neurites of N B 2 a / d l cells is in a g r e e m e n t with earlier studies in which resistant populations of microtubules are not d e t e c t a b l e early in neuronal differentiation, yet a p p e a r later in d e v e l o p m e n t 5'14'21'25. The eventual loss of nearly all neurites following e x t e n d e d periods of treatm e n t with colchicine is not u n e x p e c t e d , as all microtubules, including stabilized populations, have been shown to u n d e r g o c o m p l e t e exchange with soluble tubulin subunits within 10 h 34. Following synaptogenesis, the m o r p h o l o g y of an axon m a y r e m a i n basically unaltered t h r o u g h o u t the lifetime of the neuron; accordingly, it has been suggested that the stabilization of n e u r o n a l microtubules m a y be a key event in the establishment of m a t u r e neuronal m o r p h o l o g y 7. REFERENCES 1 Augusti-Tocco, G. and Sato, G., Establishment of functional
clonal lines of neurons from mouse neuroblastoma, Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 311-315.
2 Barra, H.S., Rodriguez, J.A., Arce, C.A. and Caputto, R., A soluble preparation from rat brain that incorporates into its own proteins [laC]arginine by a ribonuclease-sensitive system and [LaC]tyrosine by a ribonuclease-insensitive system, J. Neurochem., 20 (1973) 97-108. 3 Barra, H.S., Arce, C.A., Rodriguez, J.A. and Caputto, R., Some common properties of the protein that incorporates tyrosine as a single unit into microtubule proteins, Biochem. Biophys. Res. Comm., 60 (1974) 1384-1390. 4 Binet, S. and Meininger, V., Biochemical basis of microtubule cold stability in the peripheral and central nervous systems, Brain Res., 450 (1988) 231-236. 5 Black, M.M. and Greene, L.A., Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells, J. Cell Biol., 95 (1982) 379-386. 6 Black, M.M. and Keyser, P., Acetylation of a-tubulin in cultured neurons and the induction of a-tubulin acetylation in PC12 cells by treatment with nerve growth factor, J. Neurosci., 7 (1987) 1833-1842. 7 Black, M.M., Baas, P.W. and Humphries, S., Dynamics of a-tubulin deacetylation in intact neurons, J. Neurosci., 9 (1989) 358-368. 8 Brady, S.T., Totell, M. and Lasek, R.J., Axonal transport and axonal tubulin, J. Cell Biol., 99 (1984) 1716-1724. Burgoyne et
The d e l a y e d d e v e l o p m e n t of neurite resistance to microt u b u l e - d e p o l y m e r i z i n g drugs during differentiation of PC12 and N B 2 a / d l cells indicates that these cell lines m a y provide clues regarding the role of microtubules in axonal cytoskeletal maturation. A x o n a l neurites e l a b o r a t e d by N B 2 a / d l cells t r e a t e d with d b c A M P contain M A P 1 and tau immunoreactivity (unpublished); conversely, while M A P 2 is present in cell s o m a t a and dendritic neurites, it is a p p a r e n t l y excluded from axonal neurites 16, suggesting that inherent differences m a y exist b e t w e e n microtubules within axons, p e r i k a r y a and dendrites. T h e presence of neurofilaments 37 may also contribute to the d e v e l o p m e n t of neurite resistance to e x t e n d e d colchicine t r e a t m e n t ; since microtubules a p p e a r to cross-link with neurofilaments 2°, neurofilaments m a y further stabilize neuritic microtubules. Thus, neurite elongation m a y be directly d e p e n d e n t upon microtubule polymerization; however, it remains to be d e t e r m i n e d w h e t h e r the d e v e l o p m e n t of neurite stability is m e d i a t e d by microtubules alone, or is d e p e n d e n t u p o n the interaction of microtubule s u b p o p u l a t i o n s with o t h e r cytoskeletal constituents.
Acknowledgements. The authors wish to thank Dr. Giani Piperno (Rockefeller University, NY) for generously providing us with monoclonal antibodies 6-11B-1 and B5-1-2, and Dr. Jeanette Chlo6 Bulinski (University of California, Los Angeles, CA) for generously providing us with TYR and GLU antisera. This research was supported by the Medical Foundation, Inc./Charles A. King Trust, AG05604, and BNS-8719823.
al. 1982. 9 Cambray-Deakin, M.A. and Burgoyne, R.D., Post-translational modifications of a-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum, J. Cell Biol., 104 (1987) 1569-1574. 10 Cleveland, D.W. and Sullivan, K.E, Molecular biology and genetics of tubulin, Annu. Rev. Biochem., 54 (1985) 331-365. 11 Chiu, E-C. and Norton, W.T., Bulk preparation of CNS cytoskeleton and the separation of individual neurofilament proteins by gel filtration: dye-binding characteristics and amino acid composition, J. Neurochem., 39 (1982) 1252-1260. 12 Cohen, E., Binet, S. and Meininger, V., In situ appearance of the cold-stable microtubules in the growing axons of the tectal plate of mouse investigated immunocytochemically after polyethyleneglycol embedding, Dev. Brain Res., 36 (1987) 171-180. 13 Cumming, R., Burgoyne, R.D. and Lytton, N.A., Immunocytochemical demonstration of a-tubulin modification during axonal maturation in the cerebellar cortex, J. Cell Biol., 98 (1984) 347-351. 14 Daniels, M.P., The role of microtubules in the growth and stabilization of nerve fibers, Ann. N. Y Acad. Sci., 253 (1975) 535-544. 15 Denoulet, P., Filliatreau, G., de Nechaud, B., Gros, E and Di Giamberardino, L., Differential axonal transport of isotubulins in the motor axons of the rat sciatic nerve, J. Cell Biol., 108 (1989) 965-971. 16 Fischer, I., Shea, T.B., Sapirstein, V.S. and Kosik, K.S., Expression and distribution of microtubule-associated protein 2 (MAP2) in neuroblastoma and primary neuronal cells, Dev. Brain Res., 25 (1986) 99-109. 17 Gordon-Weeks, P.R. and Lang, R.D.A.. The ct-tubulin of the
204
18
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24 25 26 27 28 29 30
growth cone is predominantly in the tyrosinated form, Dev. Brain Res., 42 (1988) 156-160. Gundersen, G.G. and Bulinski, J.C., Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin, Eur. J. Cell Biol., 42 (1986) 288-294. Gundersen, G.G., Khawaja, S. and Bulinski, J., Postpolymerization detyrosination of a-tubulin: a mechanism for subcellular differentiation of microtubules, J. Cell Biol., 105 (1987) 251264. Hirokawa, N., Glicksman, M.A. and Willard, M.B., Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton, J. Cell Biol., 98 (1984) 1523-1536. Jacobs, J.R. and Stevens, J.K., Experimental modification of PC12 neurite shape with the microtubule-depolymerizing drug nocadazole: a serial electron microscopic study of neurite shape control, d. Cell Biol., 103 (1987) 907-915. Job, D., Rauch, C.T., Fischer, E.H. and Margolis, R.L., Recycling of cold-stable microtubules: evidence that coldstability is due to substoichiometric polymer blocks, Biochemistry, 21 (1982) 509-515. Khawaja, S., Gundersen, G.G. and Bulinski, J.C., Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level, J. Cell Biol., 106 (1988) 141-149. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 277 (1970) 680-685. Mareck, A., Fellous, A., Fracon, J. and Nunez, J., Changes in composition and activity of microtubule-associated proteins during brain development, Nature (Lond.), 284 (1980) 353-355. Miller, ED., Naus, C.C.G., Durand, M., Bloom, F.E. and Milner, R.J., Isotypes of a-tubulin are differentially regulated during neuronal maturation, J. Cell Biol., 105 (1987) 3065-3073. Murofushi, H., Purification and characterization of tubulin tyrosine ligase from porcine brain, J. Biockem., 87 (1980) 979-984. Olmsted, J., Microtubule-assoeiated proteins, Ann. Rev. Cell Biol., 2 (1986) 421-457. Olmsted, J. and Borisy, G., Characterization of microtubule assembly in porcine brain by viscometry, Biochemistry, 12 (1973) 4282-4289. Piperno, G. and Fuller, M.T., Monoclonal antibodies specific for an acetylated form of a-tubulin recognize the antigen in cilia and
31 32 33 34 35 36 37
38 39 40 41
42 43 44
flagella from a variety of organisms, J. Cell Biol., i01 (1985) 2085-2094. Piperno, G., LeDizet, M. and Chang, X., Microtubules containing acetylated a-tubulin in mammalian cells in culture, J. Cell Biol., 104 (1987) 289-302. Raybin, D. and Flavin, M., Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro, J. Cell Biol., 73 (1977) 492-504. Sale, W.S., Besharse, J.C. and Piperno, G., Distribution of acetylated a-tubulin in retina and in in vitro-assembled microtubules, Cell Motility and the Cytoskeleton, 9 (1988) 243-253. Schulze, E. and Kirschner, M., Dynamic and stable populations of microtubules in cells, J. Cell Biol., 104 (1987) 277-288. Schulze, E., Asai, D.J., Bulinski, J.C. and Kirschner, M., Post-translational modification and microtubule stability, J. Cell Biology, 105 (1987) 2167-2177. Shea, T.B., Fischer, I. and Sapirstein, V.S., The effects of retinoic acid on growth and morphological differentiation of NB2a neuroblastoma cells, Dev. Brain Res., 21 (1985) 307-314. Shea, T.B., Sihag, R.K. and Nixon, R.A., Neurofilament triplet proteins of NB2a/dl neuroblastoma: posttranslational modification and incorporation into the cytoskeleton during differentiation, Dev. Brain Res., 43 (1988) 97-109. Shelanski, M.L., Gaskin, F. and Cantor, C.R., Assembly of microtubules in the absence of added nucleotide, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 765-768. Tashiro, T., Kurokawa, M. and Komiya, Y., Two populations of axonalty transported tubulin differentiated by their interactions with neurofilaments, J. Neurochem., (1984) 1220-1225. Tashiro, T. and Komiya, Y., Stable and dynamic forms of cytoskeletal proteins in slow axonal transport, J. Neurosci., 9 (1989) 760-768. Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gets to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4350-4354. Webb, B.C. and Wilson, L., Cold-stable microtubules from brain, Biochemistry, 19 (1980) 1993-2001. Webster, D.R., Gundersen, G.G., Bulinski, J.C. and Borisy, G.G., Assembly and turnover of detyrosinated tubulin in vivo, J. Cell Biol., 105 (1987) 265-276. Yamada, K.M., Spooner, B.S. and Wessells, N.K., Axon growth: roles of microfilaments and microtubules, Proc. Natl. Acad. Sci. U.S.A., 66 (1970) 1206-1212.
195
BRESD51~8
Post-translational modification of a-tubulin by acetylation and detyrosination in NB2a/dl neuroblastoma cells Thomas B. Shea 1'2, Mary Lou Beermann I and Ralph A. Nixon t'3 1Ralph Lowell Laboratories, Mailman Research Center, McLean Hospital, Belmont, MA 02178 (U.S.A.), 2Departments of Biological Chemistry and Molecular Pharmacology and 3Program in Neuroscience, Harvard Medical School, Boston, MA 02115 (U.S.A.)
(Accepted 1 August 1989) Key words: Microtubule; a-Tubulin; Neuronal cell; Cytoskeleton; Acetylation; Detyrosination; Neuronal differentiation; Neurite maturation
Western blot analyses of total assembled microtubule fractions from NB2aJdl neuroblastoma cells demonstrated that these cells are capable of post-translationally modifying a-tubulin by acetylation and detyrosination. Immunocytochemical analyses of NB2a/dl cells differentiated with dbcAMP which had been processed under microtubule-stabilizing conditions demonstrated that all forms of a-tubulin were present throughout perikarya and neurites. By contrast, extraction of cells with Triton X-100 revealed a regional concentration of acetylated and detyrosinated a-tubulin subunits within axonal neurites, detectable in some cells after 3 days of differentiation and in nearly all cells after 7 days. Resistance of neurites to retraction following colchicine-treatment developed at a similar rate; furthermore, colchicine-resistant neurites contained intense acetylated a-tubulin immunoreactivity. We conclude that NB2a/dl cells are capable of acetylating and detyrosinating a-tubulin subunits and that selective post-translational modification of a-tubulin subunits may be related to neuritic maturation. INTRODUCTION Microtubules are thought to mediate a number of diverse cellular functions including locomotion, division, organelle and vesicular translocation, establishment and maintenance of polarity and morphology, and outgrowth and maintenance of neurites and non-neuronal processes (for review, see ref. 29). This functional diversity is achieved in part by heterogeneity in the structure of tubulin subunits, which in turn results from the existence of multiple tubulin genes 1°, and from the modification of tubulin subunits after synthesis. In addition to mediating the dynamic functions typical of most cells (above), neuronal microtubules also mediate axonal outgrowth and contribute to neuritic maintenance by interacting with the other constituents of the neuritic cytoskeleton 26"44. These diverse functions are apparently achieved in part by selective expression of tubulin isotypes and stabilization of specific microtubule subpopulations. A transient increase in the expression of m R N A s for certain a-tubulin isotypes accompanies nervous system development; this enrichment of expression is confined to regions of active neurite outgrowth 44. A similar increase in expression of m R N A coding for selective a-tubulin isotypes accompanies nerve growth factor-induced differentiation of PC12 cells 44. Subpopulations of relatively stable microtubules are concentrated
within axons 8. Axonally transported tubulin has been shown to contain a relatively slow-moving population of Triton-, cold- and colchicine-resistant microtubules which differs in subunit composition from the faster-transported tubulin p o o l 15'39'4°. Transport studies demonstrate that the slower-moving, stabilized microtubules co-migrate with neurofilaments; furthermore, when neurofilament transport was slowed by treatment with fl,fl'-iminodipropionitrile, transport of these stable microtubules was retarded to a similar extent, suggesting the presence of a physical association between neurofilaments and the stabilized microtubules 39. Several other studies have also documented the presence of cold-stable microtubule populations in n e u r o n s 4'29'42 and a regional concentration of cold-stable microtubules within axons 8'12. Cold-stable microtubules from brain contain a relatively higher concentration of two a-tubulin isotypes as compared to the overall microtubule population 22,39. Subcellular differentiation of populations of neuronal microtubules is also achieved in part by the selective post-translational modification of a-tubulin subunits by acetylation 7'31 and detyrosination 2"3'27'32. Immunocytochemical examinations of rat cerebral cortex, cerebellum, corpus callosum and brainstem demonstrated that acetyiated a-tubulin is present in neurons and is enriched in axons 9. Cultured sympathetic and granule cell neurons contain acetylated a-tubulin 6"9. The induction of acety-
Correspondence: T.B. Shea, Ralph Lowell Laboratories, McLean Hospital, Belmont, MA 02178, U.S.A.
196 l a t i o n of a - t u b u l i n has also b e e n d e m o n s t r a t e d in PC12 cells after l o n g - t e r m t r e a t m e n t with n e r v e growth factor 6. R e t i n a l m i c r o t u b u l e s c o n t a i n i n g acetylated (~-tubulin are more
resistant to d e p o l y m e r i z a t i o n by colchicine or
n o c a d a z o l e t h a n m i c r o t u b u l e s that did n o t c o n t a i n acetylated s u b u n i t s 33. T h e s e o b s e r v a t i o n s suggest that acety-
mM dibutyryl adenosine 3'.5'-cyclic monophosphate (dbcAMP; Sigma Chemical Co., St. Louis, MO) 24 h after plating. Medium was routinely changed every 3 days, with the inclusion of dbcAMP where appropriate. Untreated cells arc designated as NB2a(-): treated cells are designated as NB2a(+ 1), NB2a(+3) or NB2a(+7) to indicate the length in days of dbcAMP treatment. Cells were routinely examined and photographed by phase-contrast microscopy.
lation of a - t u b u l i n r e p r e s e n t s o n e m e c h a n i s m by which stability is c o n f e r r e d u p o n neuritic m i c r o t u b u l e s . D e t y r o s i n a t e d a - t u b u l i n s u b u n i t s are also c o n c e n t r a t e d w i t h i n n e u r i t e s 9'18. By contrast, a - t u b u l i n s u b u n i t s within m i c r o t u b u l e s in highly motile areas such as the a x o n a l g r o w t h c o n e are p r e d o m i n a n t l y t y r o s i n a t e d ~7. A l t h o u g h cold-
and
Triton-resistant microtubules
are
Colchicine treatment
Stability of neurites to colchicine was examined by incubation of cultures at 37 °C in the above medium containing 10-6 M colchicine for 2 h, after which they were examined by phase-contrast microscopy. In cases where neurites had not retracted, duplicate cultures were further incubated in the presence of colchicine for a total of 18 h.
typically
e n r i c h e d in d e t y r o s i n a t e d a - t u b u l i n s u b u n i t s , h o w e v e r , d e t y r o s i n a t i o n a l o n e is a p p a r e n t l y insufficient to confer stability to m i c r o t u b u l e s 23. Since m i c r o t u b u l e s c o n t a i n i n g significant a m o u n t s of d e t y r o s i n a t e d s u b u n i t s do n o t c o n t i n u e to e l o n g a t e 19'43, it has b e e n suggested that this s u b s e t of m i c r o t u b u l e s m a y p r e f e r e n t i a l l y be ' c a p p e d ' , a p h e n o m e n o n which confers e n h a n c e d stability a n d a l o n g e r half-life to m i c r o t u b u l e s by restricting t e r m i n a l s u b u n i t e x c h a n g e 17. A n t i b o d i e s specific for d e t y r o s i n a t e d a n d a c e t y l a t e d s u b u n i t s l a b e l e d a n identical m i c r o t u b u l e s u b p o p u l a t i o n in h u m a n r e t i n o b l a s t o m a cells, indicating that m i c r o t u b u l e s can c o n t a i n both d e t y r o s i n a t e d a n d a c e t y l a t e d s u b u n i t s ; the possibility r e m a i n s that s o m e a - t u b u l i n s u b u n i t s m a y have u n d e r g o n e both modific a t i o n s 35. M o u s e N B 2 a / d l n e u r o b l a s t o m a cells n o r m a l l y grow e x p o n e n t i a l l y a n d possess short, p u t a t i v e neurites. H o w ever, they can be i n d u c e d to e l a b o r a t e e i t h e r exclusively a x o n a l or d e n d r i t i c n e u r i t e s by t r e a t m e n t with dibutyryl cyclic A M P or r e t i n o i c acid, respectively 36. In the p r e s e n t study we d e m o n s t r a t e that m o u s e N B 2 a / d l n e u r o b l a s t o m a cells are c a p a b l e of acetylating a n d d e t y r o s i n a t i n g a - t u b u l i n s u b u n i t s . W e f u r t h e r d e m o n s t r a t e that the e l a b o r a t i o n of a x o n a l n e u r i t e s d u r i n g i n d u c e d differentiation of these cells is a c c o m p a n i e d by c o m p a r t m e n t a l ization of a c e t y l a t e d a n d d e t y r o s i n a t e d a - s u b u n i t s within cold- a n d T r i t o n - r e s i s t a n t a x o n a l m i c r o t u b u l e s ; this increase in p o s t - t r a n s l a t i o n a l l y m o d i f i e d s u b u n i t i m m u n o reactivity coincides with the a t t a i n m e n t of n e u r i t i c stability to colchicine t r e a t m e n t .
lmmunocytochemical analyses
Cultures were rinsed briefly in Tris-buffered saline (pH 7A; TBS) and fixed for 20 min with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) then rinsed 3 times with TBS. Cultures were either extracted with 1% Triton X-100 in TBS for 20 rain at room temperature or incubated under identical conditions without Triton, then incubated overnight at room temperature with mouse monoclonal antibody B5-1-2 (which reacts with all forms of a-tubulin), mouse monoclonal antibody 6-11B-1 (which reacts specifically with acetylated a-tubulin3°), rabbit polyclonal antiserum TYR (which reacts specifically with tyrosinated a-tubulin) and rabbit polyclonal antiserum GLU (which reacts specifically with detyrosinated atubulin19). The following day the cultures were rinsed 3 times in TBS (5 min each) and incubated with goat anti-rabbit or goat anti-mouse lgG conjugated with horseradish peroxidase, rinsed 3 times, and developed with diaminobenzidine in the presence of. H20 2, B5-1-2 and 6-11B-1 were generous gifts of Dr. G. Piperno, Rockefeller University, NY; TYR and GLU were generous gifts of Dr. Jeanette Chloe Bulinski, University of California, Los Angeles, CA. Isolation of total assembled microtubules and Triton-insoluble microtubules
To obtain the total assembled microtubule fraction38, cells were rinsed in TBS (30 °C), scraped from the plate with a rubber policeman in 0.1 M PIPES buffer (pH6.7) containing 1 mM MgC12, 2 mM EGTA, 0.1 mM EDTA. 2 M glycerol at 30°C and homogenized in a Dounce homogenizer at room temperature. The homogenate was centrifuged at 37.000 g for 15 rain at 30 °C. and the resulting pellet was rinsed in the same buffer and resuspended in 1% sodium dodecyl sulfate (SDS). Triton-insoluble cytoskeletons were prepared as previously described u'36. Cultures were rinsed with TBS (4 °C) then scraped from the plate with a rubber policeman in cold 1% Triton X-100 in 50 mM Tris-HCl (pH 6.8), 5 mM EDTA. 2 mM phenylmethylsulfonyl fluoride and 50/~g/ml leupeptin The cells were homogenized on ice in a Dounce homogenizer and centrifuged for 10 min at 13.000 g at 4 °C. The resulting pellet. defined as the Triton-insoluble cytoskeletal fraction, was rinsed in the same buffer without Triton. then resuspended in 1% SDS. lmmunoblot analysis
MATERIALS AND METHODS Cells and culture conditions
The NB2a/dl cell line36"37, a subclone of mouse NB2a neuroblastoma (originally derived from C1300 cells ~) was used in all experiments. Cells were plated in Dulbecco's Modified Eagle Medium containing 10% fetal calf serum and 25/~g/ml gentamycin (Gibco, Grand Island, NY) in either 10-cm plates or in multi-well chamber slides (Lab-Tek, Naperville, MD) in a humidified atmosphere of 95% air and 5% CO 2. To induce the outgrowth of axonal neurites36"37, the medium was replaced with medium containing 1
Samples were normalized with respect to total protein in the homogenate prior to fractionation, and were electrophoresed on 10% SDS gels according to Laemmli24. Nitrocellulose replicas were obtained as described41 in a Hoefer Transphor apparatus. Prestained molecular weight markers (myosin, 200 kDa; phosphorylase B, 97.4 kDa; bovine serum albumin. 68 kDa; ovalbumin. 43 kDa- achymotrypsinogen, 25.7 kDa: fl-lactoglobulin. 18.4 kDa; Bethesda Research Laboratories, Gaithersburg, MD) were included both for determination of molecular weights of immunolabeled proteins and to monitor the transfer of proteins to nitrocellulose. The proteins were visualized by sequential reaction of the replicas with 6-11B-I or B5-1-2 (1:20 dilution) or anti-GLU or anti-TYR (1:100 dilutionl
197
A B C
D
E
20097-
antibody specific for tyrosinated a-tubulin subunits (Fig. 1D), and a-tubulin subunits which had been posttranslationally modified by detyrosination (Fig. 1C) and acetylation (Fig. 1E). In each case, a single polypeptide with an apparent molecular weight of approximately 52
6643-
30Fig. 1. Characterization of anti-a-tubulin antibodies and antisera on NB2a/dl cytoskeletons, Electrophoretic analysis of total microtubule fractions of NB2a(-) cells. Lane A: representative Coomassie brilliant blue-stained gel. Lane B: nitrocellulose replica stained with monoclonal antibody B5-1-2 (specific for all a-tubulin subunits regardless of post-translationally modified state). Lane C: nitrocellulose replica stained with monoclonal antibody 6-11B-I (specific for acetylated subunits). Lane D: nitrocellulose replica stained with polyclonal antiserum TYR (specific for tyrosinated subunits). Lane E: nitrocellulose replica stained with polyclonal antiserum GLU (specific for detyrosinated subunits). The migration of molecular weight standards are noted on the left portion of the electrophoretegram. Note the immunostaining with all antibodies of a single polypeptide migrating at approximately 52 kDa.
~
~
i~
w ¸
,~ ~1~
It_
8
followed by goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (ICN Immunobiochemicals) followed by diaminobenzidine in the presence of H202 as previously described 16.
!i~iiiiii•
C V
RESULTS
Nitrocellulose replicas of the total assembled microtubule fractions from undifferentiated NB2a/dl cells were reacted with 4 different antibodies directed against a-tubulin subunits including an antibody which recognized all forms of a-tubulin subunits (Fig. 1B), an
Fig. 2. Distribution of assembled tyrosinated and detyrosinated a-tubulin subunits in NB2a/dl cells, a: phase-contrast image, b: unextracted culture stained with TYR. c: Triton-extracted culture stained with TYR. d: unextracted culture stained with GLU. e: Triton-extracted culture stained with GLU. Note that tyrosinated a-tubulin immunoreactivity is present throughout perikarya and neurites of cells before (b) and, at a reduced level, after (c) extraction. By contrast, although detyrosinated a-tubulin immunoreactivity is also present throughout perikarya and neurites of cells before (d) and after (e) extraction, only the perikaryal detyrosinated a-tubulin immunoreactivity is substantially reduced following extraction, while that of axonal neurites (arrows) is largely retained.
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198 kDa was observed, indicating that NB2a/dl cells can acetylate and detyrosinate a-tubulin subunits, and also contain a population of tyrosinated a-tubulin subunits. We next examined the intracellular distribution of a-tubulin subunits in differentiated NB2a/dl cells. Since neurons have subsets of microtubules which differ in stability, we performed immunocytochemical analyses on cells that had been processed either under conditions that stabilized all microtubules (30 °C, in the absence of Triton) or that extracted all but the subset of cold- and Triton-resistant microtubules (4 °C, in the presence of 1% Triton). Tyrosinated a-tubulin subunits were present throughout perikarya and neurites of cells harvested under microtubule-stabilizing conditions (Fig. 2a,b). Extraction of cells with Triton before immunocytochemical analyses reduced the overall level of staining; however, the extracted cells retained a similar relative distribution of tyrosinated subunit immunoreactivity (Fig. 2c). Detyrosinated subunits were also present throughout perikarya and neurites of cells processed under microtubulestabilizing conditions (Fig. 2d). However, immunoreactivity in perikarya was substantially reduced following extraction, while that of axonal neurites (arrows) underwent less reduction. Similarly, acetylated subunits were present throughout perikarya and neurites of unextracted cells (Fig. 3c); extraction of cells decreased only perikaryal immunoreactivity and did not substantially reduce acetylated a-tubulin immunoreactivity in neurites (Fig. 3d). Total a-tubulin immunoreactivity was relatively uniformly dispersed throughout perikarya and neurites in unextracted cells (Fig. 3a) and was reduced in extracted cells (Fig. 3b). The decrease in total a-tubulin immunoreactivity in neurites of extracted cells indicated that the high levels of detyrosinated and acetylated tubulin staining in neurites of similar cells does not simply reflect greater numbers of neuritic microtubules alone, but rather a selective compartmentalization of detyrosinated and acetylated subunits within cold- and Triton-resistant microtubules in neurites. Outgrowth of axonal neurites is rapid over the first 3 days following the addition of dbcAMP to NB2a/dl cells, after which neurites continue to elongate at a substantially slower rate 36'37 (see also Fig. 6). We therefore examined Triton-extracted NB2a/dl cells at several time points during differentiation to determine whether compartmentalization of acetylated and detyrosinated atubulin subunits within Triton-insoluble axonal microtubules accompanied the initial rapid phase of neurite outgrowth or was delayed. Perikaryal immunoreactivity against acetylated a-tubulin remained relatively constant throughout the first 7 days of differentiation (Fig. 4). The putative neurites of NB2a(-) cells (Fig. 4a) and the short neurites elaborated by NB2a(+I) cells (Fig. 4b) stained
at levels similar to their respective perikarya. By contrast, while a broad range of staining intensity was observed among individual neurites within each culture, some neurites of NB2a(+3) cells (Fig. 4e, small arrows) and most neurites of NB2a(+7) cells (Fig. 4g) exhibited more staining than their respective perikarya. No regional compartmentalization of total a-tubulin immu-
a
B
Fig. 3. Distribution of total and acetylated a-tubulin subunits in NB2a/dl cells, a: unextracted culture stained with B5-1-2. b: Triton-extracted culture stained with B5-1-2. c: unextracted culture stained with 6-11B-1. d: Triton-extracted culture stained with 6-11B-1. Note that immunoreactivity towards total a-tubulin is present throughout perikarya and neurites of cells before (a) and, at a reduced level, after (b) extraction. Acetytated a-tubulin immunoreactivityis also present throughout perikarya and neurites of cells before (c) and after (d) extraction; however, while perikaryal immunoreactivityis reduced followingextraction, immunoreactivity within neurites (arrows) is only slightly reduced.
199
noreactivity occurred; perikarya and their respective neurites exhibited similar levels of staining at all times during differentiation (Fig. 4b,d,f,h).
Detyrosinated a-tubulin immunoreactivity remained relatively constant within perikarya throughout differentiation (Fig. 5). The putative neurites of NB2a(-) ceils
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Fig. 4. Regional concentration of acetylated a-tubulin subunits within Triton-resistant axonal microtubules during differentiation of NB2a/dl cells. Triton-extracted cultures after 0 (a,b), 1 (b,c,), 3 (e,f) or 7 days (g,h) in the presence of dbcAMP. Panels on the left (a,c,e,g) present cultures immunostained with 6-11B-1; panels on the right (b,d,f,h) present cultures stained with B5-1-2. Note that neurites (small arrows) of day 3 and day 7 cells (e.g) contained more acetylated a-tubulin immunoreactivity than perikarya (large arrows); by contrast, total a-tubulin immunoreactivity remained evenly dispersed throughout perikarya and neurites during differentiation (b,d,f,h).
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(Fig. 5a) and the short neurites elaborated by N B 2 a ( + I ) cells (Fig. 5c) stained at levels similar to their respective perikarya. By contrast, neurites (small arrows) of N B 2 a ( + 3 ) and (+7) cells were more stained than perikarya (Fig. 5e,g, large arrows). Perikarya exhibited similar levels of tyrosinated a-tubulin immunoreactivity as their respective neurites at all times during differentiation (Fig. 5b,d,f,h).
To determine the stage when neurite stability develops in NB2a/dl cells, we examined whether or not NB2a/dl neurites developed resistance to retraction following treatment with the microtubule-depolymerizing drug colchicine. The relatively short neurites present by the first day of differentiation (Fig. 6b) were rapidly retracted by treatment with colchicine (Fig. 6c). By the 3rd day of differentiation (Fig. 6d), however, NB2a/dl
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Fig. 5. Regional concentration of detyrosinated a-tubulin subunits within Triton-resistant axonal microtubules during differentiation of NB2a/dl cells. Triton-extracted cultures after 0 (a,b), 1 (b,c), 3 (e,f) or 7 days (g,h) in the presence of dbcAMP. Panels on the left (a,c,e,g) present cultures immunostained with GLU; panels on the right (b,d,f,h) present cultures stained with TYR. Note that neurites (small arrows) of day 3 and day 7 cells (e,g) contained more detyrosinated a-tubulin immunoreactivity than perikarya (large arrows); by contrast, tyrosinated a-tubulin immunoreactivity remained evenly dispersed throughout perikarya and neurites during differentiation (b,d,f,h).
201
Fig. 6. Development of colchicine-resistance of NB2a/dl axonal neurites during differentiation. NB2a/dl cells cultures for 0 (a), 1 (b,c,), 3 (d,e) or 7 days (f,g) in the presence of dbcAMP, after which time alternate cultures were treated with colchicine (c,e,g). The short neurites of NB2a(+ 1) cells were completely retracted by treatment with colchicine (c). Neurites of day 3 cells were partially resistant to colchicine; while some neurites were completely retracted, some neurites were only partially retracted (arrows), and others seemed relatively unaffected (e). Neurites of day 7 cells were resistant to colchicine treatment (g).
neurites (Fig. 6d) had developed a degree of resistance to colchicine (Fig. 6e). Although some neurites of colchicine-treated cells had completely retracted, many had only partially retracted (Fig. 6e, arrows), and still other neurites were indistinguishable by phase-contrast microscopy from those in non-colchicine-treated N B 2 a ( + 3 ) cultures. By the 7th day of differentiation (Fig. 60, virtually all neurites were apparently unaffected by colchicine treatment as ascertained by phase-contrast microscopy (Fig. 6g). Continued exposure to colchicine for 18 h resulted in the retraction of virtually all neurites in N B 2 a ( + 3 ) cultures; however, a small percentage of
neurites in N B 2 a ( + 7 ) cultures were resistant to this extended colchicine treatment (Fig. 7b). To investigate possible mechanisms for the development of colchicine-resistance, we analyzed Triton-extracted cells for the continued presence of acetylated a-tubulin in neurites surviving colchicine-treatment. N B 2 a ( + 3 ) and (+7) cultures were treated with colchicine for 2 and 18 h, respectively, extracted with Triton, and immunostained with monoclonal antibody 6-11B-1. The Triton-insoluble cytoskeleton of colchicine-resistant neurites of N B 2 a ( + 3 ) cells (Fig. 7a) and N B 2 a ( + 7 ) cells (Fig. 7b) contained intense acetylated a-tubulin immu-
202
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Fig. 7. Colchicine-resistant neurites contain acetylated a-tubulin. Triton-extracted NB2a(+3) and (+7) cells immunostained with 6-11B-1 following treatment with colchicine for 2 (a) and 18 h (b). Note that colchicine-resistantneurites (arrows) contain more acetylated a-tubulin immunoreactivity than their respective perikarya. noreactivity, indicating that colchicine-resistant neurites contain significant amounts of acetylated a-tubulin. DISCUSSION In the present study we demonstrate that NB2a/dl cells are capable of post-translationally modifiying atubulin by acetylation and detyrosination. Subunits which had been acetylated and detyrosinated were assembled within both Triton-labile and Triton-resistant microtubules; however, these two subsets of microtubules displayed a different subcellular distribution. Triton-labile microtubules containing acetylated and detyrosinated subunits were relatively evenly distributed throughout perikarya and neurites. By contrast, Triton-resistant microtubules containing acetylated and detyrosinated subunits were concentrated within neurites of differentiated cells. This regional concentration of acetylated and detyrosinated a-tubulin subunits within axonal neurites exhibited a delayed appearance during NB2a/dl differentiation. This differential distribution was not simply a reflection of increased numbers of Triton-resistant microtubules in neurites, since extraction decreased the level of immunostaining in both perikarya and neurites by a monoclonal antibody (B5-1-2) that recognizes all forms of a-tubulin. Tyrosinated a-tubulin subunits displayed a different immunocytochemical profile in extracted cells than did subunits that had been post-translationally modified by detyrosination. Following extraction, tyrosinated immu-
noreactivity declined in both perikarya and neurites. This effect indicates that, unlike detyrosinated subunits, tyrosinated subunits are not selectively concentrated within Triton-resistant microtubules of axonal neurites, q~hese results therefore suggest that the majority of ~-tubulin subunits in Triton-resistant axonal microtubules have been detyrosinated. Similarly, the overall depletion of total a-tubulin immunoreactivity following Triton-extraction indicates that the majority of a-tubulin subunits in NB2a/dl cells exists in a Triton-soluble form, either as unassembled subunits or assembled into Triton-labile microtubules. These data are in agreement with studies in other neuronal systems, where detyrosinated a-tubulin subunits were observed to be concentrated within neurites 9'1s, while tyrosinated subunits were localized within more motile cellular areas such as growth cones tv. Regional compartmentalization of acetylated a-tubulin subunits was not detected in previous studies of cultured granule cells9, sympathetic neurons or PC12 cells°; perikarya and neurites of these cells displayed approximately equivalent levels of immunoreactivity within perikarya and neurites. It should be noted, however, that these cultures were not Triton-extracted prior to immunocytochemical analyses. Similarly, unextracted NB2a/dl cells did not exhibit regional concentrations of acetylated and detyrosinated subunits; rather, in cells harvested under microtubule-stabilizing conditions, immunoreactivity towards a-tubulin subunits which had been acetylated and detyrosinated was present throughout perikarya and neurites. Triton-extraction of NB2a/dl cells, however, revealed a selective population of stabilized axonal microtubules which were enriched in acetylated and detyrosinated subunits. It is possible that, where similar extraction procedures performed on primary neuronal cultures or PC12 cells, regional concentration of post-translationally modified a-tubulin would be observed within neurites. Neurites of NB2a(+I) cells were rapidly retracted following exposure to colchicine, while neurites of NB2a(+3) and (+7) cells developed a progressive resistance to retraction following colchicine-treatment. Since regional concentration of acetylated a-tubulin subunits within neurites was not detected until 3 days after the addition of dbcAMP, it remains a possibility that the relatively high concentration of acetylated a-tubulin in microtubules of NB2a(+3) and NB2a(+7) neurites contributes to the development of neurite stability. Previous studies, however, have indicated that acetylation and detyrosination of a-tubulin are apparently not prerequisites for microtubule stability, and that stabilization of microtubules is not necessarily preceded by subunit acetylation or detyrosination. For example, while numerous chick embryo fibroblast microtubules contain dety-
203 r o s i n a t e d a - t u b u l i n subunits, these cells possess few stable microtubules. Conversely, kidney cells of P o t o r o u s tridactylis do not contain acetylated subunits, yet possess n u m e r o u s stable microtubules. Accordingly, m o d u l a t i o n of subunit t u r n o v e r has b e e n p r o p o s e d as a m a j o r factor that underlies the d e v e l o p m e n t of stability in select microtubules35; microtubules in which subunit exchange occurs at a slower rate could therefore accumulate a g r e a t e r p r o p o r t i o n of post-translationally modified subunits, and could exhibit greater resistance to d e p o l y m e rizing t r e a t m e n t s such as cold or colchicine. In addition, differential binding of M A P s and/or interactions with o t h e r cytoskeletal constituents such as neurofilaments m a y influence microtubule stability. T h e gradual d e v e l o p m e n t of colchicine resistance in neurites of N B 2 a / d l cells is in a g r e e m e n t with earlier studies in which resistant populations of microtubules are not d e t e c t a b l e early in neuronal differentiation, yet a p p e a r later in d e v e l o p m e n t 5'14'21'25. The eventual loss of nearly all neurites following e x t e n d e d periods of treatm e n t with colchicine is not u n e x p e c t e d , as all microtubules, including stabilized populations, have been shown to u n d e r g o c o m p l e t e exchange with soluble tubulin subunits within 10 h 34. Following synaptogenesis, the m o r p h o l o g y of an axon m a y r e m a i n basically unaltered t h r o u g h o u t the lifetime of the neuron; accordingly, it has been suggested that the stabilization of n e u r o n a l microtubules m a y be a key event in the establishment of m a t u r e neuronal m o r p h o l o g y 7. REFERENCES 1 Augusti-Tocco, G. and Sato, G., Establishment of functional
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The d e l a y e d d e v e l o p m e n t of neurite resistance to microt u b u l e - d e p o l y m e r i z i n g drugs during differentiation of PC12 and N B 2 a / d l cells indicates that these cell lines m a y provide clues regarding the role of microtubules in axonal cytoskeletal maturation. A x o n a l neurites e l a b o r a t e d by N B 2 a / d l cells t r e a t e d with d b c A M P contain M A P 1 and tau immunoreactivity (unpublished); conversely, while M A P 2 is present in cell s o m a t a and dendritic neurites, it is a p p a r e n t l y excluded from axonal neurites 16, suggesting that inherent differences m a y exist b e t w e e n microtubules within axons, p e r i k a r y a and dendrites. T h e presence of neurofilaments 37 may also contribute to the d e v e l o p m e n t of neurite resistance to e x t e n d e d colchicine t r e a t m e n t ; since microtubules a p p e a r to cross-link with neurofilaments 2°, neurofilaments m a y further stabilize neuritic microtubules. Thus, neurite elongation m a y be directly d e p e n d e n t upon microtubule polymerization; however, it remains to be d e t e r m i n e d w h e t h e r the d e v e l o p m e n t of neurite stability is m e d i a t e d by microtubules alone, or is d e p e n d e n t u p o n the interaction of microtubule s u b p o p u l a t i o n s with o t h e r cytoskeletal constituents.
Acknowledgements. The authors wish to thank Dr. Giani Piperno (Rockefeller University, NY) for generously providing us with monoclonal antibodies 6-11B-1 and B5-1-2, and Dr. Jeanette Chlo6 Bulinski (University of California, Los Angeles, CA) for generously providing us with TYR and GLU antisera. This research was supported by the Medical Foundation, Inc./Charles A. King Trust, AG05604, and BNS-8719823.
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