TEKTINS AND MICROTUBULES
R. W. Linck
OUTLINE I. INTRODUCTION 11. TEKTINS IN CILIARY AND FLAGELLAR MICROTUBULES A. Fractionation of Microtubules into Protofilament Ribbons B. Fractionation of Microtubules into Tektins Filaments C. Biochemical Characterization of Tektins D. Immunological Characterization of Tektins E. Localization of Tektins in Axonemal Microtubules and Basal Bodies F. Gene Expression of Tektins in Ciliogenesis 111. SIMILARITIES BETWEEN TEKTINS AND INTERMEDIATE FILAMENT PROTEINS IV. EVIDENCE FOR TEKTINS IN CENTRIOLES AND MIOTIC SPINDLES V. CONCLUSIONS AND IMPLICATIONS ACKNOWLEDGMENTS REFERENCES Advances in Cell Biology, Volume 3, pages 35-63. Copyright 0 1990 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-013-6
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36 38 38 39 43 49 50 54 54
57 59 61 61
R. W. LINCK
36
1.
INTRODUCTION
Cilia and flagella constitute an important class of microtubules, and much can be learned from them that may be applicable to general microtubule structure and function. These organelles may be motile, such as the sperm flagella and epithelial cilia of the respiratory and reproductive tracts, or they may be nonmotile, such as primary cilia and the sensory cilia of auditory hair cells, olfactory cells, and retinal rods and cones (Barber, 1974; Dustin, 1984; Gibbons, 1981). Furthermore, the formation of primary cilia and replication of their parent centrioles are specifically timed events in the cell cycle (Bornens and Karsenti, 1986; Wilson, 1896). Finally, a number of genetic mutations and pathological conditions are known to affect the structure and function of these organelles (Eliasson et al., 1977; Huang et al., 1981; Sturgess et al., 1986). For biologists, cilia and flagella from protists and marine invertebrates have become useful systems for the study of microtubules, because it is possible to isolate and purify the “9 2” axoneme, which retains its native structure and biological activity (Gibbons, 1981). In the past few years my colleagues and I have isolated and characterized a set of novel proteins called tektins, which form extended filaments in the walls of ciliary and flagellar microtubules from invertebrates. Preliminary investigations suggested (a) that tektins were similar biochemically to the proteins forming the cytoskeletal elements known as intermediate filaments, and (b) that tektins might be associated with other microtubule systems. This paper will review the work to date on tektins from cilia and flagella and will present further evidence for their similarity to intermediate filament proteins and for the possibility that this class of proteins occurs in other microtubule systems, particularly centrioles and spindles of dividing cells. Finally, some of the possible functions of tektins will be considered.
+
Figure 1 . Fractionation of sea urchin sperm flagella: A, axonemes; B, axonemes after 6 hr dialysis against 1 mM Tris, 0.1 mM EDTA, pH 7.8; C, 0.5% Sarkosyl-insoluble ribbons of 3 protofilaments (pf-ribbons); D, 0.5% Sarkosyl/2M urea-insoluble tektin filaments. Low ionic strength (€3) disrupts the central pair microtubules, leaving relatively stable ribbons of -4 protofilaments (arrows). The Sarkosyl-insoluble pf-ribbons (C) arise primarily from doublet microtubules but some may also persist from the central-pair tubules; ribbons are composed of two morphological types: relatively bare ones (open star) and others (asterisk) which have associated material repeating axially at 16 nm (arrows). The Sarkosylhrea-insoluble tektin filaments (D) appear as individual 2 nm fibrils (small arrow) and bundles or sheets of fibrils (large arrows). Bar = 0.14 k m for A, 0.19 p m for B, 76 nm for C , 97 nm for D. A, B, and D from S. purpuratus; C from S. droebachiensis.
Tektins and Microtubules
37
R. W. LINCK
38
II. TEKTINS IN CILIARY AND FLAGELLAR MICROTUBULES A.
Fractionation of Microtubules i n t o Protofilament Ribbons
Seveial investigations provided a foundation to this work. First, Gibbons (1965) developed methods to isolate ciliary and flagellar axonemes and to purify axonemal doublet microtubules (see Figure 1). Depending on the species, doublet microtubule preparations also contain one of the central pair microtubules or remnants of the central tubules (Gibbons, 1965; Linck, 1973; Linck and Langevin, 1981; Witman et al., 1972a). Second, Stephens (1970) found that a 40°C heat treatment of sea urchin sperm flagellar doublet microtubules specifically solubilizes the B-subfibers, yielding purified singlet A-tubules; Witman et al. (1972a) devised a similar fractionation for Chlamydomonas flagella. The thermally purified A-tubules resemble their native counterpart, being composed of 13 protofilaments and an adlumenal component on the inside wall of the tubule (Linck and Langevin, 1981; Tilney et al., 1973). Finally, Meza and Witman and their colleagues reported that with either sea urchin or Chlamydomonas the extraction of axonemal microtubules with -0.5% Sarkosyl (sodium dodecyl sarcosinate) solubilizes most of each doublet microtubule, but leaves ribbons of -3 protofilaments (pfs) that are relatively stable (Meza et al., 1972; Witman, 1970; Witman et al., 1972a,b). Initially the stable 3-pf ribbon was thought to originate from the so-called “common wall” or “partition” between the A- and B-subfibers; however, subsequent electron microscopy (EM) studies indicated that the ribbon domain includes protofilaments in the region where the inner B-subfiber wall and the radial spokes make contact with the A-tubule, i.e., not the partition (Linck, 1976). Furthermore, sufficient tubulin remained in the Sarkosyl-insoluble fraction to account for possibly a second class of pf-ribbons per doublet tubule, and the central pair singlet microtubules (which don’t have partitions) were also observed to break down into relatively stable ribbons of -4 pfs (Linck, 1976; Linck and Langevin, 1982; Stephens et al., 1989). Since the loci of the ribbons are not yet precisely known, these entities will be referred to here as “stable pf-ribbons” or more specifically by the method of isolation, e.g., “Sarkosyl-insoluble pf-ribbons. The fractionation of axonemal microtubules is illustrated in Figures 1-3. The question of why pf-ribbons are so stable was intriguing and prompted further analysis (Linck, 1976). By SDS-PAGE analysis (sodium dodecyl sulfatepolyacrylamide gel electrophoresis), Sarkosyl pf-ribbons from sea urchin sperm contain reduced but still significant amounts of tubulin and are enriched in a set of polypeptides migrating somewhat faster than tubulin, plus a pair of polypeptides at -77 kd and 83 kd (Figure 2). Some of these polypeptides may account for additional material seen by negative stain electron microscopy to be regularly associated along the pf-ribbons with axial periodicities of 16 nm (Figure 1C). Although it was uncertain at the time, these additional ribbon polypeptides were ”
Tektins and Microtubules
39
regarded as non-tubulin, an assumption that has proven correct. The sum of these observations led to the proposals that the non-tubulin polypeptides participate in regulating the stability, assembly, and length of ciliary and flagellar microtubules (Linck, 1982; Linck and Langevin, 1981; Stephens, 1977). Further questions about the structure and function of the pf-ribbons and the specialized ribbon polypeptides provided the impetus for the following studies. B.
Fractionation of Microtubules into Tektins Filaments
It was found that the pf-ribbons could be further fractionated into a filamentous material of unique polypeptide composition by chaotropic agents such as KI, NaSCN, or urea (Linck et al., 1982; Linck and Langevin, 1982). By treating aliquots of sea urchin sperm flagellar axonemes with increasing concentrations of these solvents, and by analyzing the products by SDS-PAGE and EM, we could correlate certain polypeptides with the insoluble remnants of the microtubules. In this manner Wayne Vogl working in my lab examined quantitatively the fractionation of L . pictus axonemes by NaSCN (Figures 4 and 5). Typically, the 0.25M NaSCN insoluble fraction resembles the Sarkosyl-ribbon fraction, except that several high-molecular-weight polypeptides (3300 kd) also remain present, including several in the dynein region. At 0.8M NaSCN the principal polypeptides retained include those with molecular masses of -46 kd, 52 kd and 56 kd, plus two other faint bands that migrate in the positions of a-and P-tubulin, and the high-molecular-weight polypeptides. At 1.6M NaSCN the insoluble material includes primarily the 52-kd and 56-kd polypeptide bands and two polypeptides migrating in the dynein region, i.e., bands 1 and 2; the identity of these bands with respect to inner or outer dynein arms subunits is not clear (cf. Warner et al., 1989); tubulin and the 46-kd and -300-kd polypeptides are largely extracted. By EM the 0.25M NaSCN-insoluble material resembles the Sarkosyl-insoluble pfribbons, while the 1.6M NaSCN-insoluble material exists as filaments up to 6 nm in diameter and smaller fibrils 2-nm in diameter (Figure 5 ) ; the larger 6-nm filaments appear to unravel into the smaller 2-nm fibrils. Occasionally, globular particles 10 nm in diameter are seen regularly arranged along the filaments with periodicities of 2&24 nm. By quantitative gel densitometry a significant fraction of the polypeptides in the dynein region remains insoluble (Figure 6 and Table 1). Although the nature of the high-molecular-weight polypeptides has not been elucidated, they may correspond to the 1 O-nm globular particles associated with the filaments, since both the globules and the high-molecular-weight polypeptides disappear after Sarkosyl extraction. The general quantitative and qualitative results presented here are typical of the fractionation of sperm flagellar microtubules from the sea urchins Lytechinus pictus, Strongylocmtrotus droebachiensis, and Strongylocentrotus purpuratus, and from the clam Spisula solidisima; the fractionation of ciliary axonemes is also similar, but there are important differences (see below).
40
R. W. LlNCK
1
2
3
. 25
-
4
5
Tektins and Microtubules
41
Figure 3 . Cross-sectional diagrams of the doublet microtubule, showing the approximate position of one of the pf-ribbons (in black) in the A-tubule. The radial spoke side of the A-tubule is oriented down. In each model shown the pfribbon domain is associated with the “ I Ith subunit” of the B-tubule and with the shaded structure inside the A-tubule. Other types of pf-ribbon domains may be present elsewhere in the A-tubule and in central singlet microtubules. (From Linck, 1976; see also Stephens et a]., 1989). Extraction of axonemal microtubules with urea produced results similar to those above, but by combining urea with Sarkosyl, it was possible to obtain a particularly clean preparation of filaments that were free of associated particles, and to define their polypeptide composition more precisely (Linck et al., 1985; Linck and Langevin, 1982; Linck and Stephens, 1987). As shown in Figure ID, a two-fold extraction of flagellar microtubules with 0.5% Sarkosyl and 2M urea yields a filamentous preparation composed of fibrils 2 nm in diameter; the fibrils often seem to be aggregated into sheets and bundles. In S . purpuratus this filamentous material consists of three major polypeptide bands at 47 kd, 51 kd and 55 kd, present in equimolar amounts; proteins migrating in the positions of tubulin or dynein are not seen on overloaded gels (Figure 2). Higher concentrations of urea, KI, or NaSCN tend to solubilize the 47-kd polypeptide band (Figure 4), but a filamentous residue still remains; in particular, 3M in 0.5% Sarkosyl urea cleanly extracts the 47-kd polypeptide, leaving equimolar quantities of the 51-kd and 55-kd species. On the basis of these observations, it was proposed that these residual proteins were related and that they formed the filaments (Linck, 1982; Linck et al., 1982). Because of their fibrous appearance and presumed structural role in microtubules, the filamentous proteins were Figure 2. Electrophoretograms (SDS-PAGE) of fractionated flagellar doublet microtubules from S.purpuratus: lanc I , doublet microtubules; lanes 2 and 3 , 0.5% Sarkosyl-insoluble pf-ribbons; lane 4 and 5 , tektin filaments after one and two extractions respectively with 0.5% Sarkosyli2M urea. Sarkosyl pf-ribbons are composed of residual a(a)- and P(b)-tubulin and associated proteins (M,X 1000). Filaments are composed of 3 principal tektins, A (55-kd), B (51-kd) and C (47-kd); a weaker band is usually present at -53 kd. Lane pairs 1-2 and 3-4 are loaded stoichiometrically to reflect the quantitative recovery of proteins in the insoluble fractions. (From the author’s work and from molecular mass data from Chang, 1987. Compare with Table 4.)
R. W. LlNCK
42
Dynein 1
4-1 -2
2
Tubulin
Tektin
a.
+A
P-
Ax
.5Ax
.2Ax
Axonernes
.25
.35
.45
55
.80
t
B
t
C
1.60
NaSCN (M)
Figure 4. SDS-PAGE, showing the fractionation of L . pictus axonemes by NaSCN. To demonstrate linearity between protein loading and staining intensity, contr2l axonemes (Ax) were loaded at three concentrations (lx = 25 kg/lane). Identical amounts of axonemes, relative to the lx control, were extracted with the indicated concentrations of NaSCN or with 0.15M NaCl for Ax controls. Samples were then centrifuged at lo5 X g for 90 min; pellets were dissolved in equal volumes of SDS media, dialyzed, and applied to gel Lanes in equal volumes. Gels were stained with 0.0175% Serva Blue in 25% isopropanol, 10%
Tektins and Microtubules
43
Figure 5 . Negative stain electron micrograph of the 1.6M NaSCN-insoluble fraction from L . pictus, shown in Figure 4. This fraction consists of extended fibrils 2 nm in diameter (small arrow) and bundles of fibrils (large arrow). Globular particles are frequently seen arranged along the fibrils with periodicities of 2 G 2 4 nm (arrowheads). Mag bar = 150 nm.
given the name “tektins” (from the Greek tektonos, meaning architecture, builder), reviving an early term used by Mazia (1968) in reference to cytoskeletal and microtubule proteins. The polymers composed of these proteins are tentatively referred to as tektin filaments. C.
Biochemical Characterization of Tektins
Although we do not presently know how many different tektins there are, recent efforts have concentrated on characterizing the tektins obtained by the 0.5% Sarkosyli2M urea extraction of flagellar microtubules. Two sea urchin species have been closely examined. The molecular masses appear to be different acetic acid, and destained in 10% acetic acid; with this procedure, protein staining (i.e., optical density) is linear with protein loading (see Figure 6). At 0.8M NaSCN the insoluble fraction consists primarily of three tektins (A, B, and C) and polypeptides migrating in the dynein region (bands 1 and 2), as quantitated in Table 1 . These residual high-molecular-weight proteins may correspond to inner or outer dynein arms subunits (cf. Sale et al., 1989; Warner et al., 1989).
R. W. LINCK
44
A
1
B
1
t
0
0
~
0.8 M NaSCN
Figure 6. Densitometric quantitation of the high-molecular-weight polypeptides remaining in the NaSCN-insoluble fractions from L . pictus. (A), JoyceLoebl densitometer tracings from corresponding lanes of Figure 4, top of gel to left; note, peak areasiheights of dynein in control axonemes (Ax) are linear with loading concentration. (B), tracing from a higher resolution, 3% acrylamide gel for the 0.8M NaSCN sample. For comparison purposes, the areas of residual bands I and 2 were measured from 3% gels and plotted as a percent of the major dynein bands 1 and 2 respectively, in control axonemes; see Table 1 . Table I . Percentages of High-molecular-weight Proteins Remaining in the NaSCN-insoluble Fraction* Bund I
Barid 2
Exp. I
E.rp. 2
S%
E.tp. I
Exp. -7
1%
0.35 M
16.0
15.8
15.9
12.5
13.3
12.9
0.80 M
15.3
13.7
14.5
10.8
10.0
10.4
NnSCN
*The dynein regions of 3% acrylamide SDS gels were scanned (see Fig. 66). using a Joyce Loebl Microdenaitometer. The areas of residual bands I and 2 were expressed as a percentage of the major dynein bands I and 2 respectively. in axoneme controls.
Tektins and Microtubules
45
Table 2. Comparative Molecular Masses and Immunological Relatedness of Tektins* Apparent Molecular Mass. !iD L . pictus
S . purpurutus
56-57
55
B
51-52
51
C
46
47
Tekrirr
A
*The tektins are arranged in three groups. A. B. and C. in descending order of apparent molecular mass, based on comparative SDS-PAGE. Antibodies to each of the tektins (anti-tektins) are primarily monospecific within a species. and anti-tektins from one species strongly cross-react with only the same tektin type in the other species. Thus. the tektins are categorized by similarities in molecular mass and immunological cross-reactivities. Note that anti-tektin C cross-reacts weakly with tektin A within the same apecies (see Fig. 7 ) as well as between species (not shown). (Reprinted with permission from Steffen and Linch, 1988)
for the two species, as determined by SDS-PAGE in side-by-side comparisons on slab gels. In our hands with Bio Rad Electrophoresis Grade SDS, tektins from S. purpurutus have apparent masses of 47 kd, 5 1 kd and 55 kd, while those from L . pictus measure 46 kd, 5 1-52 kd, and 5 6 5 7 kd (see Table 2) (Steffen and Linck, 1988). With BDH brand SDS, Chang and Piperno (1987) reported masses for S. purpuratus tektins of 46 kd, 49 kd, and 53 kd. It is not presently known whether the differences in the measurements for S. purpuratus tektins reflect standard experimental error, or whether the purity of SDS affects the apparent mass (perhaps even the order of migration) of the tektins, as has been found with the tubulins (Bibring et al., 1976). The important point, however, is that the molecular masses of the tektins vary slightly between species; positive identification of corresponding tektins is possible by immunological methods (see below). The composition of the tektin filaments has also been analyzed by 2-D isoelectric focusing (IEF)/SDS-PAGE (Figure 8) and is described below. The major Sarkosyllurea-insoluble tektins from S . purpuratus have been characterized biochemically in collaborative studies with Raymond Stephens (Linck and Stephens, 1987). The 47-kd, 51-kd, and 55-kd tektins and also a-and P-tubulins were purified by SDS-PAGE and compared by high-resolution 2-dimensional tryptic peptide mapping and amino acid analysis. Tryptic peptide mapping reveals a 6 3 4 7 % coincidence in the number and position of peptides from the 5 I-kd tektin, as compared to the 47-kd and 55-kd tektins, and a >70% coincidence between the 47-kd and 55-kd tektins; none of the tektins, however, bears any significant degree of similarity with either tubulin. The amino acid
46
R. W. LINCK
12345
Figure 7. SDS-PAGE immunoblot characterization of affinity-purified, polyclonal rabbit antibodies to L . pictus tektins (anti-tektins). Lane 1 , electrophoretogram of 0.5% SarkosyU2M urea-insoluble tektin filaments stained with Serva Blue. Nitrocellulose, strips were stained with anti-tektin C (lane 2), anti-tektin B (lane 3), anti-tektin A (lane 4), and Amido Black (lane 5 ) . The anti-tektins are primarily monospecific, with anti-tektin C cross-reacting weakly but consistently with tektin A. Reprinted with permission from Steffen and Linck (1988); for complete characterization of the anti-tektins, see Linck et al. (1987).
compositions of the tektins are also similar to each other, but very different from the tubulins (Table 3). Three equimolar tektins can also be separated from each other and from tubulin by reverse-phase HPLC on an acetonitrile gradient in trifluoroacetic acid; their elution profiles are on the order of 5 1 kd, 55 kd, and 47 kd, which correspond to their relative hydrophobicities, as predicted from their amipo acid compositions. These results conclusively demonstrated that the tektins are distinct but related polypeptides. By all criteria the tektins are different from tubulin, but by amino acid compositions they are strikingly similar to intermediate filament proteins (Table 3; detailed similarities and differences are reviewed later). It should be noted here that the 47-kd and 55-kd tektins are each characterized by a predominant spot on 2-D IEF-SDS-PAGE gels (see Fig.
Tektins and Microtubules
47
Tuble 3. Amino Acid Compositions (mole %) of Tektins from S . purpurutus and Comparison with Intermediate Filament Proteins Tektins' Residue
Ala Arg Asxl Cy Glx' GIY
His Ile
Leu Lys
Met Phe Pro Ser Thr TrP TY r Val
A
6.48 7.85 16. I 1.27 17.0 5.23 0.67 4.44 9.61 7.03 2.67 1.54 2.03 4.99 5.98 1.24 1.56 3.99
< <
< >
>
>
8.10 7.38 14.1 1.99 17.2 6.05 1.17 3.73 9.17 7.01 1.16 1.37 2.63 6.16 6.75 1.19 1.79 3.09
> >
<
< <
<
Itirerrnediate Filottierit Proteir~s~'
C
Average
Destnirz
Vimrririri
Keratin
7.41 6.55 13.3 2.01 16.2 5.52 1.26 3.38 9.02 8.02 2.67 2.27 2.56 5.82 7.05 1.10 1.81 4.08
7.45 7.26 14.5 1.76 16.8 5.6 1.03 3.85 9.27 7.35 2.17 1.73 2.41 5.66 6.59 1.18 1.72 3.72
9.07 9.50 8.64 0.22 20.09 3.67 I .73 4.32 9.50 4.54 2.59 2.81 I .73 6.70 6.05 0.22 3.02 5.62
6.68 9.27 I I .64 0.22 19.18 3.02 I .29 3.45 12.07 4.74 I .94 2. I6 I .72 9.27 5.17 0.22 2.80 5.17
4.37 5.46 8.51 0.87 15.07 21.18 0.22 3.28 10.48 3.71 1.31 2.84 0.66 10.92 3.71 0.22 3.93 3.28
B
('From Linck & Stephens (1987). I'Calculated from Weber & Geisler (1985)
< and > denote major differences among tektins Aax' combines A m and Asp: Glx' combines Glu and Gln Cys and Trp: important differences between tektins and IF proteins.
8 and Table 4 of next section), while the 51-kd tektin resolves into two closely spaced, approximately equal spots. No attempt was made to separate the two 51kd polypeptides for amino acid analysis and peptide mapping, but it does not seem likely that the basic conclusions of this characterization would be affected by possible heterogeneity of the 5 1-kd tektin. Other investigations indicate that tektins are fibrous proteins. Filaments prepared from S. purpurutus by Sarkosylhrea extraction and composed predominantly of the 55-kd and 51-kd tektins have -70% a-helix, as measured by circular dichroism (Linck and Langevin, 1982); Xiao-jia Chang (1987) in Gianni Piperno's laboratory obtained a somewhat lower value of 55-60% for the same two tektins (with the molecular weight differences as noted abovej. Sarkosylurea filaments from S. purpuratus containing equimolar quantities of the 47-kd, 5 I-kd, and 55-kd tektins were also studied by Lorena Beese (1984) in Carolyn Cohen's laboratory, using x-ray diffraction. These preparations could easily be pulled into fibers, which yielded strong a-type patterns. The observed a-helical structure of the tektins is also consistent with their low proline contents (Linck
48
R. W. LINCK
Figure 8. One-dimensional SUS-PAGE (left) and two-dimensional IEF/SDSPAGE (right) elcctrophoretograms of 0.5% Sarkosyl-insoluble pf-ribbons (fop) and 0.5% SarkosyU2M urea-insoluble tektin filaments (bottom) from S. purpuratus. Compare with Table 4. lsoelectric values are indicated along the top. and molecular masses (kd) are given along the 1-D SDS gel to the left. 2-D immunoblots of either the pf-ribbons or the tektin filaments show identical results, namely: antibody to tektin A (55-kd) stains spots a, b, and c; antibody to tektin B (51-kd) stains spots a' and b'; and antibody to tektin C (47-kd) stains spots a", b", c", and d", and cross-reacts weakly with the 55-kd spot a. From Linck et al. (1987) and Steffcn and Linck ( 1 9 8 9 ~ )the ; MI.and pl values diffcr somewhat from those reported by Chang (1987) and Chang and Piperno (1987). and Stephens, 1987) and with the observation that tektins contain large polypcptidc domains, resistant to proteolytic degradation (Chang and Piperno, 1987). According to Cohen ( 1966; Cohen and Parry, 1986; and personal communication), these data identify the tektins as a-type proteins.
Tektins and Microtubules
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Table 4. Molecular Masses and Isoelectric Points of Major Polypeptides Comprising the Sarkosyl-resistant Protofilament Ribbons of S. purpuratus Sperm Flagellar Microtubules" Componentb
Isoelectric point
83-kD ( .1 ) 77-kD ( J ) a ,-tubulin az-tubulin P-tubulin 55-kD-a 5S-kD
53-kD 5 I-kD-a' 5 I-kD-b' 51-kD-c' 47-kD-C" 38-kD 35-kD 34-kDc 25-kD 25-kD
6.75 6.50 5.85 5.70 5.60 6.90 >8 6.10 6.20 6.10 6.00 6.15 n.d n.d n.d >8 -8
Tektin filaments are primarily composed of tektin A (55 kD-a), tektin B (51 kD-a' and 51 k-D-b'). and tektin C (47kD-c"); a 53-kD polypeptide is present in lesser amounts. ,'From Linck et al. (1987). except as noted. ',For identification. see Figs. 2 and 8. 'From Chang (1987); see Fig. 2 here. The 55-kD component was given as 53-kD by Chang; since it co-migrates with tektin A. its mass has been adjusted to conform to the masses given here.
D.
immunological Characterization of Tektins
The tektins have been further characterized by immunological methods. Polyclonal antibodies have been raised against each tektin from two sea urchin species and used in characterization and localization studies (Linck et al., 1987; Steffen and Linck, 1988, 1989a,b). Three tektins from L . pictus and three from S. purpuratus were purified by SDS-PAGE. Rabbit antibodies against each tektin (anti-tektins) were raised and affinity-purified, using SDS-denatured tektin
R. W. LINCK
50
filaments from L. pictus as the affinity probe. By immunoblot analysis, as shown in Figure 7, each anti-tektin was primarily specific for its own antigen in the homologous species and cross-reacted strongly with only one tektin in the heterologous species. Consequently, even though the molecular weights of tektins differ between L. pictus and S. purpurutus, the anti-tektins are tektintype-specific; i.e., antibody against a given tektin from one species primarily recognizes the same tektin-type in a different species. Thus, by immunological and molecular weight criteria, we have identified three tektin types (Table 2): tektins A (55-57-kd), B (51-52-kd), and C (46-47-kd). These anti-tektins crossreact with apparently related polypeptides in cilia, both from sea urchin and molluscs, but the pattern of tektins is somewhat more complex (Linck et al., 1987; Stephens et al., 1989). Tektin filaments and the Sarkosyl-insoluble pf-ribbons from S. purpurutus have also been analyzed by 2-D IEFISDS-PAGE and 2-D immunoblotting, as described in Figure 8 and Table 4 (Linck et al., 1987). Chang (1987) has reported several additional polypeptides and somewhat different PI values for tdktins compared to ours; these details are noted in Figure 2 and Table 4. By immunoblotting analysis with our affinity-purified polyclonal anti-tektins, the pf-ribbon and tektin filament fractions give identical results: namely, anti-tektin A stained primarily a 55-kd/pI 6.9 spot, anti-tektin B primarily two 51-kd/pl -6.15 spots, and anti-tektin C primarily one 47-kd/pI 6.15 spot; as in 1-D immunoblots, anti-tektin C weakly but consistently stained the major 55-kd (pl 6.9) spot. Anti-tektins A and C also stained several satellite spots shifted by -0.1 PI units. At present we assume that the major polypeptide spots (55 kd-a, 5 1 kd-a’, 5 1 kd-b’, and 47kd-c”) correspond to the major tektins polypeptides. The other neighboring polypeptides may be isoelectric variants, novel tektins, or unrelated contaminants copurifying with the antigens. By a similar approach tektins have been studied by Chang and Piperno (1987), using monoclonal antibodies. The monoclonal antibodies presently characterized include: antibody 1-4-2 specific for tektin A, antibody 1-17-1 specific for tektin B, and antibodies 3-7-1 and 3-10-1 specific for tektin C. These antibody probes have been useful for studying tektin polypeptide structure, localization, and similarities to intermediate filament proteins, as discussed elsewhere in this chapter. E.
Localization of Tektins in Aaxonemal Microtubules and Basal Bodies
Several determinations regarding the localization of tektins have been made using immunological methods. In collaborative studies with Linda Amos and W. B. Amos, affinity-purified, polyclonal antibodies were prepared against whole tektin filaments composed of all three tektins (Amos et al., 1985, 1986; Linck et al., 1985); for this work the antigens were isolated with Sarkosyhrea and not denatured by SDS. The antibodies recognized the three major tektins on immu-
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noblots, and by immunofluorescence microscopy the anti-tektins stained sperm flagella and embryonic cilia from several species of sea urchins, as well as cilia from molluscan gill tissue. In preparations for immunofluorescence microscopy the antibodies stained methanol-fixed axonemes (i.e., microtubules), but in EM experiments the antibodies did not appear to significantly label unfixed, native microtubules, implying that the antigenic sites of the tektins are normally buried in the microtubule wall or masked by associated proteins but are exposed by fixation or disruption of the tubule (Figure 9A). In fact, if microtubules are attached to carbon film EM grids and then extracted on the grid surface with Sarkosylhrea to solubilize tubulin, filaments appear which label with anti-tektins (Figure 9B). These results suggest that the tektins are distributed along the length of the microtubules and strongly imply that the tektins exist as filaments in the microtubule wall, as opposed to aggregating artificially into filaments during the extraction of tubulin. In support of the interpretation that tektins pre-exist as filaments in the microtubule, the anti-tektins are also seen to label thin fibrils projecting from the ends of protofilament ribbons (Amos et al., 1986; Linck et al., 1985); see Figure 9. More recent immunolocalization studies have employed the affinity-purified, polyclonal antibodies specific for each tektin, as described earlier (Linck et al., 1987; Steffen and Linck, 1988). These antibodies were also found to recognize ciliary and flagellar tektins from a variety of species. In immunofluorescence studies of L . pictus sperm, Walter Steffen found that the axonemes splay into their nine individual doublet microtubules and used this phenomenon to determine the composition of tektins in each tubule. As represented in Figure 10, each anti-tektin stains the entire lengths of nine filaments, as does anti-tubulin; the central-pair microtubules are not preserved in this species, as judged by their lack of anti-tubulin staining. These results indicate that tektins A, B, and C are all present in each doublet tubule and A-tubule extension, assuming of course that the specificities of the antibodies in the immunofluorescence staining procedure are essentially the same as in the SDS-PAGE immunoblot assay. In an effort to examine the possible presence of tektins in the central pair, we examined another species, the bat star, Patiria miniuta. In this case two filaments were faintly stained with anti-tubulin in addition to the nine doublet tubules, suggesting that the central-pair tubules, or more likely their remnants, were preserved; these two filaments were also faintly stained with anti-tektin C, suggesting the presence of tektin(s) in the central singlet microtubules (cf. Steffen and Linck, 1988). Tektins have also been localized by independent immuno-biochemical approaches. First, Chang (1987) used monoclonal antibodies to demonstrate that in S. purpurutus tektins A, B, and C are associated exclusively with the Sarkosylinsoluble pf-ribbons of axonemal microtubules; none of those tektins were found in the Sarkosyl-soluble fraction by immunoblotting techniques. Second, Stephens et al. (1989) found that ciliary axonemes from both molluscan gill tissue and sea urchin embryos can be fractionated by a 40°C heat treatment or by
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Figure 9. Localization of tektins in sperm flagellar microtubules by negative stain immuno EM. A and B from S . purpurutus: (A) -sample obtained from low ionic strength dialysis-treated axonemes, incubated with rabbit antibodies to whole tektin filaments, followed by 5 nm gold-conjugated goat anti-rabbit IgG; note, there is little, if any, labeling along the axis of the microtubules or pf-
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Figure 10. Localization of tektins in L. pictus sperm by irnmunofluorescence microscopy. Sperm are attached to cover slips such that the axonemes splay apart. Treatment with each anti-tektin (in the example shown, anti- L . pictus tektin B) reveals a similar staining pattern: nine filaments attached at their proximal end to the intensely stained basal body (to the left). Similar patterns of splayed filaments are obtained with anti-tubulin, indicating that the nine filaments correspond to the doublet tubules and that the central pair tubules are not preserved. Mag bar = 10 km. (From Steffen and Linck, 1988.) Sarkosyllurea into a “skeleton” of the former axoneme, consisting of nine outer filamentdpf-ribbons surrounding one or two central filamentslpf-ribbons. Using the polyclonal anti-tektins of Linck et al. (1987), Stephens and colleagues found that antigens recognized by anti-tektins A and B were associated almost entirely with the filamentous fraction of the axoneme; only anti-tektin C revealed partial solubilization of tektin antigens. Finally, tektins or tektin-like components have been localized to basal bodies. As illustrated in Figure 10, all three polyclonal anti-tektins display intense immunofluorescence staining of the basal bodies in sea urchin sperm (Steffen and Linck, 1988). In addition, monoclonal antibody 3-7-1 to tektin C crossreacts on immunoblots with -4650-kd polypeptides in basal bodies isolated from Chlamydomonas and judged to be free of contaminating axonemes (Chang, 1987). These results are not entirely surprising, given that tektins are present in ribbons. (B) parallel sample attached to EM grid and pretreated with 0.5% SarkosyV2M urea before antibody staining; filaments remain which label with anti-tektins (note gold particles). C-F from P . miliaris: anti-tektins are consistently found to label thin fibrils -2 nm in diameter extending from the ends of pf-ribbons (arrows), also seen in A . Mag bar = 194 nm for A-B , 100 nm for CF. (From Linck et al., 1985.)
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axonemal A-tubules and that the A-tubules of axonemes and basal bodies are continuous structures. F.
Gene Expression of Tektins in Ciliogenesis
As components of cilia and flagella, tektins might be expected to be important in the development of these organelles. In earlier investigations of ciliary regeneration in S. droebuchiensis sea urchin embryos, Stephens (1977) reported that, following deciliation, regenerated cilia contain a polypeptide (“Component 20,” M,55 kd) that is synthesized de novo in limited amounts, as determined by pulse/ chase experiments; additional synthesis of this polypeptide is required in the second round of regeneration. Recent experiments demonstrate that the loss of this component after regeneration is not due to protein turnover but to complete utilization and incorporation into the ciliary axoneme (Stephens, 1989). Component 20 had been previously shown to be one of the major polypeptides of the pfribbon domain of A-tubules (Linck, 1976), and it has now been shown to be recognized by our antibody to the 55-kd tektin A from S. purpurutus (Stephens, 1989, Stephens et al., 1989). In related studies Chang (1987) used monoclonal antibodies to correlate the presence of tektins with ciliogenesis. In Chang’s studies the levels of tektins B and C are weakly detected in unfertilized eggs but increase sharply after fertilization, whereas tektin A is not detected until the blastula stage when ciliogenesis occurs. Finally, Jan Norrander in my lab and in collaboration with Ray Stephens is currently examining the appearance of tektin mRNA, using cDNA probes; her preliminary results suggest that tektin A gene expression is largely coupled to ciliogenesis (Norrander et al., 1988). From these collected studies the expression of tektin A, rather than the majority of other, preexisting axonemal proteins, would seem to be of paramount importance to the assembly of cilia and flagella. Stephens (1977) originally postulated that the quantal, de novo synthesis of Component 20, now 55-kd tektin A, was consistent with it being a factor involved in the elongation and/or length determination of ciliary microtubules. Our antibody labelling, showing that the 55-kd tektins (as well as tektins B and C) are present through the length of flagellar doublet microtubules (Steffen and Linck, 1988), is at least consistent with Stephens’ hypothesis. In any event, the close association of tektins with tubulin to form the pf-ribbon would seem to implicate tektins in microtubule assembly in cilia, flagella and presumably centrioles and basal bodies.
111.- SIMILARITIES BETWEEN TEKTINS AND INTERMEDIATE FILAMENT PROTEINS Based on initial observations, David Albertini, I and our colleagues noted similarities between sea urchin tektins and mammalian intermediate filament (IF) proteins (Linck et al., 1982). Both sets of proteins are insoluble in less than 5 M
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urea; they share molecular masses in the range of 46 kd to 60 kd; their isolelectric points cluster around 6.0 and 6.8; they possess greater than 60% a-helix; and finally, unless the filamentous state of tektins is a preparation artifact, tektin filaments and IFs are composed of -2 nm subfibrils or protofibrils. It is perhaps appropriate first to brietly’review the molecular structure of IF subunits and polymers. Several different IF proteins have been characterized: keratins, desmin, vimentin, glial fibrillary acid protein, and neurofilament proteins (Goldman et al., 1986; Steinert and Roop, 1988; Wang et al., 1985). Most IF subunits form homopolymers but can also form heteropolymers, while keratins are obligate heteropolymers. By thin section EM, the filaments’ diameters in vivo are reported to range from 7 to 8 nm for some types of IFs to 12 nm for neurofilaments. Although heterogeneous in molecular weight and immunological properties, IF proteins have a common subunit structure: each is composed of an a-helical rod domain of -37 kd and random coil N- and C-terminal domains. The a-helical rod domains of the different IF proteins have several important properties: they form the backbone of the intermediate filament, and they share strong primary sequence homologies, being characterized by a heptapeptide repeat, a-b-c-d-e-f-g,where a and d a r e non-polar residues (Geisler and Weber, 1982; Hanukoglu and Fuchs, 1982; Steinert et al., 1983). Such a sequence leads to the production of an a-helix, consistent with the observed a-type x-ray patterns observed for 1Fs (Fraser et a]., 1972; Steinert et al., 1978). The globular domains are variable in sequence and molecular mass, extend from the filament backbone, and presumably provide functional properties to the native IF (Albers and Fuchs, 1987). The current model indicates that the filament backbone is composed of approximately 16 subunits, which are arranged axially as 8 protofibrils of coiled coils, 2 nm in diameter (Aebi et al., 1983; Steven et al., 1985). Recently, nuclear lamins A and C have been found to have close primary sequence homology to the a-helical rod domain of IF proteins (Fisher et al., 1986; McKeon et al., 1986), and although the morphology of the lamina in vivo is different from that of IFs, isolated lamins can form IF-like structures in vitro (Aebi et a]., 1986). At this writing we still do not know the true relationship between tektins and IF proteins, and we can only point out a number of additional similarities and some differences that have recently come to light. First, using a monoclonal antibody against tektin filaments, Amos et al. (1986) detected an -48 nm periodicity along the filament axis; this and similar structural repeats have been measured for IFs (Ip et al., 1985; Steven et al., 1985). This repeat corresponds theoretically to an -37-kd domain composed mainly of a-helix. In support of this model Chang and Piperno (1987) have shown that tektin C can be cleaved by chymotrypsin to yield fragments similar in size to the a-helical rod domain of IF subunits. Amino acid analysis of tektins by Linck and Stephens (1987) revealed a striking similarity to IF proteins (see Table 3), with residue differences between IF proteins being sometimes greater than the differences between a particular IF protein and a tektin. Low proline contents are noteworthy and consistent with
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large a-helical domains. Chang and Piperno ( 1987) developed two monoclonal antibodies to tektin C and studied their cross-reaction with IF proteins by immunofluorescence and immunoblot analysis: one monoclonal (3-7- I ) crossreacts with desmin and vimentin, and the other (3-10-1) cross-reacts with nuclear lamins A and C. While the cross-reaction of a monoclonal antibody might be fortuitous, Walter Steffen in my laboratory has found that polyclonal antibodies raised against three different tektins cross-react with specific keratins by immunofluorescence and immunoblot analysis; see Figure 11 (Steffen and Linck, 1989b). The anti-tektins also appear to stain the nuclear envelope in different vertebrate cells (Edson et al., 1987; Steffen and Linck, 1989a). Finally, Jan Norrander in my lab has sequenced approximately two thirds of tektin A by cDNA methods; according to her preliminary results (manuscript in preparation), tektin A is predicted to be highly a-helical and is similar to keratins and nuclear lamins; while this sequence correlation is low, it is strengthened by the observation that polyclonal antibody to tektin A cross-reacts with keratins (Steffen and Linck, 1989b). It is important as well to point out several differences between tektins and IFs. Structurally, the filaments differ in size: IFs have a dense core diameter of -9 nm, which presumably corresponds to the filamentous assembly of rod domains (Steinert et al., 1985; Steven et al.. 1983); the actual lower limit of IF diameters
Figure 11. Phase contrast and immunofluorescence images of pig kidney LLCPK, cells stained with affinity-purified antibody to L . pictus tektin B, revealing a pattern of filaments strikingly similar to that of cytokeratins. Antitektin B also cross-reacts with specific keratins on immunoblots. Mag bar = 25 km. See Steffen and Linck (1989b).
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in vivo, however, is not known. The maximum possible diameter of tektin filaments is not known either, but is not likely to be greater than -6 nm; it is also not yet clear whether tektins exist in the form of these larger filaments or as smaller 2 nm fibrils in the microtubule wall. The amino acid compositions also reveal two potentially important differences: IF proteins contain a single tryptophan at a conserved site, and desmin and vimentin likewise posses a single conserved cysteine. On the other hand, tektins possess multiple cysteines and tryptophans. In spite of these differences the similarities between tektins and IFs are intriguing, and current sequencing efforts will hopefully resolve this issue.
IV.
EVIDENCE FOR TEKTINS IN CENTRIOLES AND MITOTIC SPINDLES
A final topic of this paper concerns whether tektins are unique to cilia and flagella or whether they or related proteins are fundamental to other microtubule systems. The staining of basal body components of cilia and flagella with monoclonal anti-tektins (Chang, 1987) and polyclonal anti-tektins (Steffen and Linck, 1988) suggested that tektins might also be present in centrioles. This expectation is strongly supported by immunofluorescence microscopy, showing that centrioles from a variety of mammalian cell lines are stained by polyclonal antibodies to sea urchin tektins (Steffen and Linck, 1988, 1989a). Our results were obtained using affinity-purified anti-tektins and their Fab fragments, the antibodies were used at the same low concentrations suitable for staining sea urchin sperm, and the centriole staining could be eliminated in preabsorption control experiments. The presence of tektins in centrioles implied by these collected results are not entirely surprising, given that tektins appear to be linear components of ciliary and flagellar A-tubules and that the A- and B-tubules assemble from their parent triplet microtubules in centrioles and basal bodies. Evidence linking tektins to the spindles of dividing cells is also becoming clearer. It is well known that isolated meiotic and mitotic apparati retain their poles, astral fibers, spindle fibers, and chromosomes (cf. Salmon, 1982); spindles from sea urchin and clam are composed principally of tubulin and associated proteins, in particular a 55-kd polypeptide (Hays and Salmon, 1983; Pratt et al., 1980; Rebhun and Palazzo, 1986, 1987). When isolated spindles are subjected to agents that depolymerize microtubules, a remnant or matrix of the spindle remains, which retains the original fibrous appearance and spindle shape; this matrix is significantly depleted of tubulin and is composed largely of the -55-kd polypeptide (Hays and Salmon, 1983; Rebhun and Palazzo, 1986, 1987). According to Rebhun (personal communication), the amino acid composition of the 55-kd polypeptide is strikingly similar both to vertebrate IF proteins and to sea urchin tektins. Furthermore, preliminary investigations in sea urchins by Chang and Piperno indicate that monoclonal antibody 3-7- I to flagellar tektin C crossreacts in immunoblots with a non-flagellar 48-kd polypeptide retained in a 1%
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Sarkosyl-insoluble extract of isolated spindles; however, the cross-reaction was not observed by immunofluorescence microscopy, perhaps because of problems with fixation or the masking of the antigen with tubulin and microtubuleassociated proteins (Chang, 1987; Chang and Piperno, 1983). Finally, my colleagues and I have investigated the cross-reaction of the polyclonal antitektins with spindle components. Initial studies also failed to show a cross-reaction in sea urchins (Amos et a]., 1985), but in these experiments the antibodies were prepared against non-SDS denatured tektin filaments and the antibodies might not recognize the determinants of even a related spindle protein. Working with the more recently developed antibodies to the individual SDS-purified tektins (Linck et al., 1987), Walter Steffen and I have demonstrated by irnmunofluorescence and immuno EM that affinity-purified antibodies to tektin A, B , or C recognize components in mitotic spindles and midbodies of a variety of cell types-see Figure 12 (Steffen and Linck, 1987, 1989a); interestingly, some antitektins that label spindles also cross-react with IFs and IF proteins, as determined by inmunoblot and immuno EM methods. A possibly related observation has been made by Raymond et al. (1987), showing that an antibody raised against an
Figure 12. Phase contrast and immunofluorescence images of a dividing LLCPK, cell stained with affinity-purified antibody to S. purpururus tektin C. Anti-tektins stain the poles and both diffuse and filamentous components of the spindle; the filaments correspond to phase-dense structures. Anti- S. purpurutus tektin C also shows a faint, diffuse fluorescence of the cytoplasm. Mag bar = 10 p.m. (From Steffen and Linck, unpublished).
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-50-kd high salt insoluble cytoplasmic protein from unfertilized sea urchin eggs stains the mitotic spindle in sea urchins.
V.
CONCLUSIONS AND IMPLICATIONS
Structural Organization and Functions of Jektins in Microtubules
The major unresolved questions about tektins concern their structural organization and function(s) in microtubules. The structure of tektins in microtubules has not been completely elucidated, but for reasons given in this paper, the working hypothesis is that the tektins form a-helical coiled coils that extend throughout the length of a given microtubule. The evidence from cilia and flagella so far suggests that three tektins, tektins A, B, and C, are integral components of a relatively stable 3-protofilament ribbon domain in the A-tubules and possibly in central-pair singlet microtubules; other tektins may exist in these and other microtubule systems. Within the pf-ribbon it is not clear whether the presumed filaments of tektin exist as small fibrils, e.g., 2-3 nm in diameter, located between grooves of tubulin protofilaments, or whether tektins assemble to form one or more of the 13 protofilaments of the microtubule wall. The latter possibility is particularly intriquing, considering the amount of tektin present in the pf-ribbon: the molar ratio of tubulin ( a + p) to tektins (A + B C) to the 77/83-kd polypeptide pair is estimated to be approximately 4 : 2 : 1 (Linck, unpublished observations). This estimate is in close agreement with the model in which two of the three protofilaments in the pf-ribbon domain are composed of tubulin and the other protofilament is composed of tektins, with the 77/83-kd proteins associated periodicially along the ribbon axis, such as in Figure 1C. The determination of tektins as either fibrils or protofilaments will affect the eventual classification of these proteins. In the event that they are 2-3 nm fibrils between tubulin protofilaments, tektins could be regarded as intrinsic microtubuleassociated proteins (MAPS), i.e., those that can only be solubilized by disruption of the microtubule; whereas if tektins actually form protofilaments, they would be more properly regarded as true microtubule-proteins as with tubulin. Our understanding of microtubule structure derives from the optical diffraction studies of Amos and Klug (1974) on flagellar doublet microtubules. From that work the B-tubule was shown to have an arrangement of tubulin dimers defined as the B-lattice; the A-tubule was more complex and said to be composed of a different arrangement of dimers, the A-lattice. Several lines of evidence have since suggested that microtubules are composed predominantly of the B-lattice (Linck and Langevin, 1981; Linck et al., 1981; Mandelkow et al., 1977, 1986; Mandelkow and Mandelkow, 1989; McEwen and Edelstein, 1980; Woodrum and Linck, 1980); however, x-ray diffraction studies have not yet directly demonstrated the existence of the B-lattice (Beese et al., 1987; Cohen et al., 1971; Mandelkow et al., 1977; Wais-Steider et al., 1987). The significance for a
+
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microtubule with a B-type lattice is that the tubule would contain at least one helical discontinuity or “seam” between two protofilaments (cf. Linck and Langevin, 1981; Mandelkow et al., 1986). Since the seam represents a sterospecifically unique point around the tubule wall, and since the tektin-tubulin pf-ribbon is a chemically unique domain of the microtubule, then it seems probable that these two entities are one in the same, i.e., the association of tubulin with putative tektin filaments forms the seam. Regardless of the exact structural organization of tektins (fibrils versus protofilaments), their intimate and likely direct association with tubulin predicts they will influence microtubule structure, assembly, and function. Although the functions of tektins are largely unknown, there are several distinct possibilities for their role in cilia and flagella and other microtubule systems, as outlined below (cf. Linck, 1989). 1. The formation or preexistence of a stable tubulin-tektin filament complex could be a key element in the nucleation of microtubule assembly. A putative seam in the tubulin lattice could specify the site of tektin assembly; conversely, tektins could direct the formation of the seam. 2. The interaction of tektin and tubulin within the pf-ribbon could determine the angle between adjacent protofilaments, and thereby govern the number of protofilaments assembled into a microtuble. 3. Tektins may regulate microtubule length, according to the hypothesis of Stephens (1977). This idea has emerged from experiments clearly showing that ciliary microtubule length during sea urchin ciliogenesis is limited by the amount of tektin A synthesized (Stephens, 1989). The amount of tektin available for assembly may in turn be regulated transcriptionally, as our preliminary evidence would suggest (Norrander et al., 1988). 4. The tight association between tektin, tubulin, and the 77/83-kd polypeptides seems a likely basis for the stability of the pf-ribbon. In conjunction with other proteins and physiological events, the tubulin-tektin filament complex might function in stabilizing certain classes of microtubules (cf. Behnke and Forer, 1967). This idea is supported in part by the association of tektins with the most stable classes of microtubules, i.e., cilia, flagella, basal bodies, centrioles, and possibly with midbody microtubules (Steffen and Linck, 1988, 1989a). 5 . Finally, tektins may code for 3-dimensional spatial information along microtubules. The position(s) of tektins around the tubule wall and the long-range periodicities that may be provided by a helically structured tektin filament could provide binding sites for various axonemal components. In this regard preliminary evidence points to the association of tektin filaments with dynein-like polypeptides (Figures 4-6 and the discussion above) and with nexin filaments and radial spoke components (cf. Amos et al., 1976; Stephens et al., 1989).
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In conclusion, continued studies of tektins are likely to elucidate the functions of these proteins in cilia, flagella, basal bodies, and centrioles. More generally, it will now be important to determine whether tektin-like proteins with similar functions are present in other microtubule and intermediate filament systems.
ACKNOWLEDGMENTS The author gratefully acknowledges the stimulating interactions and collaborations with David Albertini, Linda and W.B. Amos, Lorena Beese, Carolyn Cohen, Jan Norrander, Walter Steffen, Ray Stephens and Wayne Vogl. This work has been supported by U.S. P.H.S. grants GM-21527 and GM-35648 and N.S.F. grant DCB-8811015 to the author.
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