Turnover of tubulin in ciliary outer doublet microtubules

Previous pulse-chase labeling studies have shown that structural proteins incorporate into fully assembled sea urchin embryonic cilia at rates approaching those of full regeneration. When all background ciliogenesis was suppressed by taxol, the turnover of most proteins, including tubulin, continued. The present study utilized chemical dissection to explore the route of tubulin incorporation in the presence of taxol and also in steady-state cilia from prism stage embryos. Surprisingly, in cilia from untreated embryos, the most heavily labeled tubulin was found in the most stable portion of the doublet microtubles, the junctional protofilaments. With taxol, this preferential incorporation was suppressed, although control-level turnover still took place in the remainder of the doublet. This paradoxical result was confirmed by pulse-chase labeling and immediately isolating steady-state cilia, then isolating two additional crops of cilia regenerated, respectively, from pools of high and then decreased label. In each case, the level of label occurring in the tubulin from the junctional protofilaments, compared with that from the remainder of the doublet, correlated with the level of pool label from which it must exchange or assemble. These data indicate that ciliary outer doublet microtubules are dynamic structures and that the junctional region is not inert. Plausible mechanisms of incorporation and turnover of tubulin in fully-assembled, fully-motile cilia can now be assessed with regared to recent discoveries, particularly intraflagellar transport, distal tip incorporation, and treadmilling.     

The in vitro assembly of flagellar outer doublet tubulin

Flagellar outer doublet microtubules were solubilized by use of sonication, and the tubulin was reassembled in vitro into single microtubules containing 14 and 15 protofilaments. The tubulin assembly was dependent on both the KCl and tubulin concentrations, exhibiting a critical concentration of 0.72 mg/ml at optimum solvent conditions. Flagellar tubulin was purified by cycles of temperature-dependent assembly-disassembly and molecular sieve chromatography, and characterized by two-dimensional gel electrophoresis. Although doublet microtubules were not formed in vitro, outer doublet tubulin assembled onto intact A- and B-subfibers of outer doublet microtubules and basal bodies of Chlamydomonas; the rate of assembly from the distal ends of these structures was greater than that from the proximal ends. Microtubule-associated proteins (MAPs) from mammalian brain stimulated outer doublet tubulin assembly, decorating the microtubules with fine filamentous projections.     

Preferential incorporation of tubulin into the junctional region of ciliary outer doublet microtubules: a model for treadmilling by lattice dislocation

Even in the presence of colchicine or Taxol(R), sea urchin embryonic cilia undergo substantial steady-state turnover, with a rate of tubulin incorporation approaching half that seen in full regeneration [Stephens: Mol Biol Cell 8:2187-2198, 1997]. Preliminary experiments suggest that tubulin incorporates differentially into the most stable portion of the outer doublet, the junctional protofilaments [Stephens: Cell Struct Funct 24:413-418, 1999]. To explore this possibility further, embryos of the sea urchin Tripneustes gratilla, a ciliary length inducible system [Stephens: J Exp Zool 269:106-115, 1994a], were pulse labeled with (3)H leucine during steady-state turnover or induced elongation, followed by regeneration in the presence of unlabeled leucine. Cilia were isolated by hypertonic shock and fractionated into detergent-soluble membrane plus matrix, thermally-solubilized microtubule walls, and insoluble 9-fold symmetric remnants of A-B junctional protofilaments plus associated architectural elements. The fractions were resolved by SDS-PAGE and the specific activity of alpha-tubulin was determined. In cilia undergoing turnover or elongation during an isotope pulse, the specific activity of tubulin in the junctional region approximated that of precursor membrane plus matrix tubulin but surpassed that of the tubule wall by a factor of approximately 1.5. In cilia regenerated during an isotope chase, the specific activity of junctional tubulin exceeded that of both the membrane plus matrix and the tubule wall by a similar factor. These data indicate that tubulin is preferentially incorporated into junctional protofilaments during steady-state turnover, induced elongation and regeneration. A model for directional incorporation based on surface lattice discontinuities in the outer doublet is proposed.     

In vitro polymerization of microtubules from HeLa cells

Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.     

Functional protofilament numbering of ciliary, flagellar, and centriolar microtubules

This article discusses the current state of knowledge about the evolutionarily conserved structure of ciliary, flagellar and centriolar microtubules, and formally proposes a functional numbering convention for their protofilaments.     

In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs

The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28 degrees C. In homogenates of unactivated eggs few microtubules form and no asters are observed. By 2.5 min after activation microtubules polymerize in association with a dense central cylinder, resulting in the formation of small asterlike structures. By 4.5 min after activation the asters formed in vitro contain a distinct centriole, and microtubules now radiate from a larger volume of granular material which surrounds the centriole. By 15 min (metaphase I) the granular material is more disperse and only loosely associated with the centriole. Microtubules are occasionally observed which appear to radiate directly from one end of the centriole. The organizing center can be partially isolated by centrifugation of homogenates of metaphase eggs and will induce aster formation if mixed with tubulin from either activated or unactivated eggs. Pretreatment of the eggs with colchicine does not prevent the formation of a functional organizing center. Complete spindles can also be obtained under polymerizing conditions by either homogenizing the eggs directly into warm buffer or by adding a warm high-speed supernate to spindles which have been isolated in a microtubule stabilizing medium. Extensive addition of new tubulin occurs onto the isolated spindles, resulting primarily in growth of astral fibers, although there occasionally appears to be growth of chromosomal fibers and of pole-to-pole fibers. Negatively stained aster microtubules have a strong tendency to associate side by side, and under some conditions distinct cross bridges can be observed. However, under other conditions large numbers of 300-400-A particles surround the microtubules; the presence of stain between particles can give the appearance of cross bridges.     

Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.     

Cyclical interactions between two outer doublet microtubules in split flagellar axonemes

The beating of cilia and flagella is based on the localized sliding between adjacent outer doublet microtubules; however, the mechanism that produces oscillatory bending is unclear. To elucidate this mechanism, we examined the behavior of frayed axonemes of Chlamydomonas by using high-speed video recording. A pair of doublet microtubules frequently displayed association and dissociation cycles in the presence of ATP. In many instances, the dissociation of two microtubules was not accompanied by noticeable bending, suggesting that the dynein-microtubule interaction is not necessarily regulated by the microtubule curvature. On rare occasions, association and dissociation occurred simultaneously in the same interacting pair, resulting in a tip-directed movement of a stretch of gap between the pair. Based on these observations, we propose a model for cyclical bend propagation in the axoneme.     

Head-to-tail polymerization of microtubules in vitro

The kinetics of microtubule polymerization to steady-state and the ability of tubulin subunits to exchange with polymer at steady-state were examined to determine the applicability of the head-to-tail polymerization mechanism (Wegner, 1976) to microtubule assembly in vitro. Under conditions where self-nucleation was a rare event, tubulin was induced to polymerize by the addition of short microtubule fragments, and the kinetics of elongation were analyzed as a pseudofirst-order reaction. At steady-state, a trace amount of [³H]tubulin, prepared by labeling in vivo of chick brain protein, was added to polymerized microtubules and the kinetics of label uptake into polymer were monitored by a rapid centrifugal assay. The isotope exchange kinetics were analyzed according to a theoretical model previously applied to actin polymerization (Wegner, 1976) and extended for the case of microtubule polymerization. The rate of head-to-tail polymerization, expressed as the steady-state subunit flux, was 27·6 ± 7·6 per second at 37 °C. The head-to-tail parameter s, a measure of the efficiency of subunit flux, was 0·26 ± 0·07, indicating that four association and four dissociation events resulted in the flux of one subunit through the polymer at steady-state.The role of GTP in this mechanism of microtubule polymerization was examined by replacement of the nucleotide occupying the exchangeable binding site of tubulin with the non-hydrolyzable GTP analog guanosine 5′-(β,γ-methylene)triphosphate. It was found that the rate of steady-state flux was reduced by two orders of magnitude compared to tubulin polymerized with GTP. The head-to-tail parameter approached its limiting value of zero, indicating greatly reduced efficiency of subunit flux through the polymer in the presence of this analog.In summary, this study demonstrates that microtubules exhibit significant headto-tail polymerization in the presence of GTP and, in keeping with theoretical considerations, provides evidence that nucleotide hydrolysis is required for subunit flux through the polymer.     

Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly

B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication- derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.     

Virions and membrane proteins of the potato X virus interact with microtubules and enables tubulin polymerization in vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.     

In vitro biosynthesis of rat sperm outer dense fiber components

Conditions were established for in vitro culture of seminiferous tubules of adult rat testis. Tubules fragments were able to incorporate radioactive amino acids for up to 6 hours of incubation at 32 degrees C in a modified Eagle's minimum media, indicating biosynthetic activity. Addition of D-glucose (11 Mm) increased the incorporation of either [3H] Leucine or [35S] Methionine four-fold in the protein components of seminiferous tubules. Polyclonal antibodies against outer dense fibers (ODF) polypeptides, which represent approximately 30% of the total sperm proteins, immunoprecipitated 5% of the total radioactivity from cultures carried out either in the presence or absence of D-glucose. Moreover, antibodies specific for the 27-30 kilodalton polypeptides of ODF immunoprecipitated 2% of the total radioactivity, showing no differences in the presence and absence of D-glucose. This study indicates that ODF polypeptides can be synthesized in vitro at 32 degrees C with and without D-glucose.     

Characterization and in vitro polymerization of Tetrahymena tubulin

Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.     

Sulfhydryls and the in vitro polymerization of tubulin

The free sulfhydryls of brain tubulin prepared by cyclic polymerization procedures both with and without glycerol have been examined. The average free sulfhydryl titer of tubulin prepared with glycerol (7.0 sulfhydryls/55,000 mol wt) is greater than that of tubulin prepared without glycerol (4.0 sulfhydryls/55,000 mol wt). Diamide, a sulfhydryl- oxidizing agent, inhibits the polymerization of tubulin. Diamide also disperses the 20S and 30S oligomers of tubulin seen in analytical ultracentrifuge patterns of tubulin solutions and, depending on the temperature at which diamide is added, converts all or part of the oligomeric material to 6S dimers. Electron microscopy demonstrates that diamide also destroys the 450-A ring structures characteristic of tubulin solutions. All diamide effects are reversible by the addition of 10 mM dithioerythreitol, a sulfhydryl-reducing agent. That diamide interacts with sulfhydryls on tubulin is directly demonstrated by a 50% decrease in the free sulfhydryl titer of tubulin measured after diamide treatment. Concentrations of CaCl2 which inhibit polymerization also decrease the free sulfhydryl titer of tubulin.     

Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control.

A central question in cell biology is how cells determine the size of their organelles. Flagellar length control is a convenient system for studying organelle size regulation. Mechanistic models proposed for flagellar length regulation have been constrained by the assumption that flagella are static structures once they are assembled. However, recent work has shown that flagella are dynamic and are constantly turning over. We have determined that this turnover occurs at the flagellar tips, and that the assembly portion of the turnover is mediated by intraflagellar transport (IFT). Blocking IFT inhibits the incorporation of tubulin at the flagellar tips and causes the flagella to resorb. These results lead to a simple steady-state model for flagellar length regulation by which a balance of assembly and disassembly can effectively regulate flagellar length.