Flagellar microtubule dynamics in Chlamydomonas: cytochalasin D induces periods of microtubule shortening and elongation; and colchicine induces disassembly of the distal, but not proximal, half of the flagellum

To study the mechanisms responsible for the regulation of flagellar length, we examined the effects of colchicine and Cytochalasin D (CD) on the growth and maintenance of Chlamydomonas flagella on motile wild type cells as well as on pf 18 cells, whose flagella lack the central microtubules and are immobile. CD had no effect on the regeneration of flagella after deflagellation but it induced fully assembled flagella to shorten at an average rate of 0.03 microns-min. Cells remained fully motile in CD and even stubby flagella continued to move, indicating that flagellar shortening did not selectively disrupt machinery necessary for motility. To observe the effects of the drug on individual cells, pf 18 cells were treated with CD and flagella on cells were monitored by direct observation over a 5-hour period. Flagella on control pf 18 cells maintained their initial lengths throughout the experiment but flagella on CD-treated cells exhibited periods of elongation, shortening, and regrowth suggestive of the dynamic behavior of cytoplasmic microtubules observed in vitro and in vitro. Cells behaved individually, with no two cells exhibiting the same flagellar behavior at any given time although both flagella on any single cell behaved identically. The rate of drug-induced flagellar shortening and elongation in pf 18 cells varied from 0.08 to 0.17 microns-min-1, with each event occurring over 10-60-min periods. Addition of colchicine to wild type and pf 18 cells induced flagella to shorten at an average rate of 0.06 microns-min-1 until the flagella reached an average of 73% of their initial length, after which they exhibited no further shortening or elongation. Cells treated with colchicine and CD exhibited nearly complete flagellar resorption, with little variation in flagellar length among cells. The effects of these drugs were reversible and flagella grew to normal stable lengths after drug removal. Taken together, these results show that the distal half to one-third of the Chlamydomonas flagellum is relatively unstable in the presence of colchicine but that the proximal half to two-thirds of the flagellum is stable to this drug. In contrast to colchicine, CD can induce nearly complete flagellar microtubule disassembly as well as flagellar assembly. Flagellar microtubules must, therefore, be inherently unstable, and flagellar length is stabilized by factors that are sensitive, either directly or indirectly, to the effects of CD.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

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.     

Microtubule dynamics in interphase cells

The sites of microtubule growth and the kinetics of elongation have been studied in vivo by microinjection of biotin-labeled tubulin and subsequent visualization with immunocytochemical probes. Immunofluorescence and immunoelectron microscopy demonstrate that injected biotin-labeled subunits are incorporated into new segments of growth which are contiguous with unlabeled microtubules. Rapid incorporation occurs by elongation of existing microtubules and new nucleation off the centrosome. The growth rate is 3.6 micron/min and is independent of the concentration of injected labeled tubulin. This rate of incorporation together with turnover of existing microtubules leads to approximately 80% exchange in 15 min. The observed kinetics and pattern of microtubule turnover allow for an evaluation of the relevance of several in vitro models for steady-state dynamics to the in vivo situation. We have also observed a substantial population of quasi-stable microtubules that does not exchange subunits as rapidly as the majority of microtubules and may have specialized functions in the cell.     

Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration

Flagellar regeneration in gametes of Chlamydomonas reinhardi is initiated within 15–20 min after flagellar amputation and proceeds at a rapid but decelerating rate until by 90 min flagellar outgrowth is 80–85% complete. Sufficient flagellar protein reserves exist in the cytoplasm to allow regeneration of flagella $\text{1}\text{3}\text{–}\text{1}\text{2}$ normal length. Nevertheless, in vivo labeling with ¹⁴C-amino acids shows that microtubule protein and other flagellar proteins are synthesized de novo during flagellar regeneration. To determine whether tubulin is synthesized continuously by gametic cells or whether its synthesis is induced as a consequence of deflagellation, we have isolated polyribosomes from deflagellated and control cells, and analyzed the proteins produced by these polyribosomes during in vitro translation. Two proteins of 53,000 and 56,000 molecular weight which co-migrate with flagellar and chick brain tubulin on SDS-polyacrylamide gels and which selectively co-assemble with chick brain tubulin during in vitro microtubule assembly are synthesized by polyribosomes (or polyadenylated mRNA) from deflagellated cells. No microtubule proteins can be detected in the translation products synthesized by polyribosomes (or mRNA) from control cells, clearly indicating that deflagellation results in the induction of tubulin synthesis.Kinetics of tubulin synthesis demonstrate that induction takes place immediately after deflagellation; polyribosomes bearing tubulin mRNA can be detected in the cytoplasm in as little as 15 min after removal of flagella. Maximal rates of tubulin synthesis occur between 45 and 90 min after deflagellation when approximately 14% of the protein being synthesized by the cell is tubulin. This estimate of tubulin synthesis based on in vitro translation data agrees well with in vivo measurements of flagellar tubulin synthesis. While high levels of tubulin production extend well beyond the period of rapid flagellar assembly, synthesis begins to decline after 90 min, and by 180 min after deflagellation only low levels of tubulin mRNA are detectable in polyribosomes.     

Microtubule Assembly and Function in Chlamydomonas: Inhibition of Growth and Flagellar Regeneration by Antitubulins and Other Drugs and Isolation of Resistant Mutants

The distribution of microtubules in Chlamydomonas reinhardtii suggests that they are involved in mitosis, cell and nuclear cleavage, and generation of flagella. Vinblastine, colchicine, and podophyllotoxin bind to the protein building block of microtubules (tubulin) and prevent normal assembly. Mutants resistant to these "antitubulin" drugs are candidates to have alterations in tubulin primary structure. We report the ability to inhibit growth, and flagellar regeneration after amputation, of: vinblastine, several colchicine derivatives, two water-soluble derivatives of podophyllotoxin (succinylpodophyllotoxin and epipodophyllotoxin thiuronium bromide), and other substances which may interfere with flagellar assembly or motility (isopropyl N-phenyl carbamate, 2-methoxy-5-nitrotropone, chloral hydrate, caffeine, and nickel acetate). The ability of each drug to inhibit binding of labeled colchicine or podophyllotoxin to mammalian brain tubulin was also determined. The results suggest that only in the cases of colchicine, colcemide, and epipodophyllotoxin thiruonium bromide was the toxicity to Chlamydomonas mediated by inhibition of tubulin assembly. The requirement for high concentrations of colchicine may be due to permeability barriers, since colchicine toxicity was potentiated by deoxycholate. Mutants resistant to antitubulins were isolated after treatment with methyl methanesulfonate. The results with vinblastine were equivocal. Of three mutants resistant to inhibition of growth and flagellar regeneration by colchicine, one was also cross-resistant to epipodophyllotoxin thiuronium bromide.     

Polarity of flagellar assembly in Chlamydomonas.

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.     

Dynamics of Antarctic fish microtubules at low temperatures

The tubulins of Antarctic fishes, purified from brain tissue and depleted of microtubule-associated proteins (MAPs), polymerized efficiently in vitro to yield microtubules at near-physiological and supraphysiological temperatures (5, 10, and 20 degrees C). The dynamics of the microtubules at these temperatures were examined through the use of labeled guanosine 5'-triphosphate (GTP) as a marker for the incorporation, retention, and loss of tubulin dimers. Following attainment of a steady state in microtubule mass at 20 degrees C, the rate of incorporation of [3H]GTP (i.e., tubulin dimers) during pulses of constant duration decreased asymptotically toward a constant, nonzero value as the interval prior to label addition to the microtubule solution increased. Concomitant with the decreasing rate of label incorporation, the average length of the microtubules increased, and the number concentration of microtubules decreased. Thus, redistribution of microtubule lengths (probably via dynamic instability and/or microtubule annealing) appears to be responsible for the time-dependent decrease in the rate of tubulin uptake. When the microtubules had attained both a steady state in mass and a constant length distribution, linear incorporation of labeled tubulin dimers over time occurred at rates of 1.45 s-1 at 5 degrees C, 0.48 s-1 at 10 degrees C, and 0.18 s-1 at 20 degrees C. Thus, the microtubules displayed greater rates of subunit flux, or treadmilling, at lower, near-physiological temperatures. At each temperature, most of the incorporated label was retained by the microtubules during a subsequent chase with excess unlabeled GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants

Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.     

Regulation of flagellar length in Chlamydomonas

Chlamydomonas reinhardtii has two apically localized flagella that are maintained at an equal and appropriate length. Assembly and maintenance of flagella requires a microtubule-based transport system known as intraflagellar transport (IFT). During IFT, proteins destined for incorporation into or removal from a flagellum are carried along doublet microtubules via IFT particles. Regulation of IFT activity therefore is pivotal in determining the length of a flagellum. Reviewed is our current understanding of the role of IFT and signal transduction pathways in the regulation of flagellar length.     

An essential quality control mechanism at the eukaryotic basal body prior to intraflagellar transport

Constructing a eukaryotic cilium/flagellum is a demanding task requiring the transport of proteins from their cytoplasmic synthesis site into a spatially and environmentally distinct cellular compartment. The clear potential hazard is that import of aberrant proteins could seriously disable cilia/flagella assembly or turnover processes. Here, we reveal that tubulin protein destined for incorporation into axonemal microtubules interacts with a tubulin cofactor C (TBCC) domain-containing protein that is specifically located at the mature basal body transitional fibres. RNA interference-mediated ablation of this protein results in axonemal microtubule defects but no effect on other microtubule populations within the cell. Bioinformatics analysis indicates that this protein belongs to a clade of flagellum-specific TBCC-like proteins that includes the human protein, XRP2, mutations which lead to certain forms of the hereditary eye disease retinitis pigmentosa. Taken with other observations regarding the role of transitional fibres in cilium/flagellum assembly, we suggest that a localized protein processing capacity embedded at transitional fibres ensures the 'quality' of tubulin imported into the cilium/flagellum, and further, that loss of a ciliary/flagellar quality control capability may underpin a number of human genetic disorders.     

Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.     

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.     

Assembly of flagellar radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3

Radial spokes of the eukaryotic flagellum extend from the A tubule of each outer doublet microtubule toward the central pair microtubules. In the paralyzed flagella mutant of Chlamydomonas pf14, a mutation in the gene for one of 17 polypeptides that comprise the radial spokes results in flagella that lack all 17 spoke components. The defective gene product, radial spoke protein 3 (RSP3), is, therefore, pivotal to the assembly of the entire spoke and may attach the spoke to the axoneme. We have synthesized RSP3 in vitro and assayed its binding to axonemes from pf14 cells to determine if RSP3 can attach to spokeless axonemes. In vitro, RSP3 binds to pf14 axonemes, but not to wild-type axonemes or microtubules polymerized from purified chick brain tubulin. The sole axoneme binding domain of RSP3 is located within amino acids 1-85 of the 516 amino acid protein; deletion of these amino acids abolishes binding by RSP3. Fusion of amino acids 1-85 or 42-85 to an unrelated protein confers complete or partial binding activity, respectively, to the fusion protein. Transformation of pf14 cells with mutagenized RSP3 genes indicates that amino acids 18-87 of RSP3 are important to its function, but that the carboxy-terminal 140 amino acids can be deleted with little effect on radial spoke assembly or flagellar motility.     

Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body

Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.     

Kinesin-like proteins in the flagella of Chlamydomonas

The flagella of the biflagellate unicellular alga Chlamydomonas have long been known to contain the microtubule-dependent motor protein dynein, but recent findings indicate they also contain multiple members of the kinesin superfamily. Two of these kinesin-like proteins are restricted to a single central-pair microtubule, raising the question of how proteins are targeted to specific microtubules within the flagellum. The kinesin-like proteins on the central-pair microtubules may cause the central-pair apparatus to rotate or twist during flagellar beating. Other kinesins within the flagellum may participate in movements associated with the flagellar membrane.     

Tetrahymena tubulins and in vitro translation of Tetrahymena RNA.

Tetrahymena outer doublet tubulin was compared with neurotubulin and Chlamydomonas flagellar tubulin on SDS-polyacrylamide gels. Tetrahymena alpha tubulin did not comigrate with either brain or flagellar alpha tubulins, although brain, flagellar, and ciliary beta tubulins all comigrated. Axonemal tubulin from Tetrahymena strain ST was compared with this tubulin from strains W, S, HSM, and E, and all were found to have the same mobilities. Poly-A containing RNA was separated from whole cell Tetrahymena RNA by oligo-dT cellulose chromatography. Poly-A+ RNA from 24-h cultures (early exponential growth) stimulated greater incorporation of amino acids into polypeptides in the wheat germ cell-free translation system than did poly-A+ RNA from 36-h and 49-h cultures. When separated on SDS-polyacrylamide gels, the translation products of the 24-h poly-A+ RNA had 2 prominent protein bands which comigrated with alpha and beta tubulin isolated from Tetrahymena cilia. These bands were not found in the translation products of poly-A+ RNA isolated from 49-h cultures or in the translation products of poly-A- RNA.     

Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly

We previously reported that direct incorporation of GDP (i.e., without an initial hydrolysis of GTP) into microtubules occurs throughout an assembly cycle in a constant proportion. The exact proportion varied with reaction conditions, becoming greater under all conditions in which tubulin-GDP increased relative to tubulin-GTP (low Mg2+ and GTP concentrations, high tubulin concentrations, and in the presence of exogenous GDP). These findings led us to explore further interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly. We have now determined the minimum amount of tubulin-GTP required for the initiation of microtubule assembly and the relative efficiency with which tubulin-GDP participates in microtubule elongation. When GTP, GDP, and tubulin concentrations were varied at a constant Mg2+ concentration (0.2 mM), initiation of assembly required that 35% of the nucleotide-bearing tubulin be in the form of tubulin-GTP, and incorporation of tubulin-GDP into microtubules during elongation was only 60% as efficient as would be predicted on the basis of its proportional concentration in the reaction mixtures. Very different results were obtained when the Mg2+ concentration was varied. Even though Mg2+ enhances the binding of GTP to tubulin (the equilibrium constant for the exchange of GTP for GDP was 0.2 in the absence of exogenous Mg2+, 3 with 0.2 mM Mg2+, 5 with 0.5 mM Mg2+, and 11 with 2 and 4 mM Mg2+), as Mg2+ was increased the proportion of tubulin-GTP required for the initiation of microtubule assembly rose greatly, and the direct incorporation of tubulin-GDP into microtubules during elongation became progressively more efficient. In the absence of exogenous Mg2+, only 20% tubulin-GTP was required for initiation, and tubulin-GDP was directly incorporated into microtubules half as efficiently as would be predicted on the basis of its concentration in the reaction mixture. At the highest Mg2+ concentration examined (4 mM), 80% tubulin-GTP was required for initiation of assembly, and tubulin-GDP was incorporated into microtubules as efficiently as tubulin-GTP.     

The Microtubule plus end-tracking protein EB1 is localized to the flagellar tip and basal bodies in Chlamydomonas reinhardtii

Flagellar axonemes assemble and continuously turn over at the flagellar tip. The supply and removal of axonemal subunits at the tip are mediated by intraflagellar transport (IFT), a motility process essential for the assembly and maintenance of all eukaryotic flagella and cilia. IFT is characterized by the movement of large protein complexes (IFT particles) from the basal bodies to the flagellar tip by kinesin-II and from the tip back to the basal bodies by cytoplasmic dynein 1b. The IFT particles consist of approximately 16 polypeptides partitioned into two complexes, A and B, and associate with axonemal precursors/turn over products. The mechanisms by which IFT motor regulation and cargo loading/unloading occur at the flagellar tip are unknown. We identified a Chlamydomonas reinhardtii ortholog of the microtubule (MT) plus end-tracking protein EB1 [4] (CrEB1) and show here that CrEB1 localizes to the tip of flagella and to the proximal part of the basal bodies. Furthermore, we found that CrEB1 is depleted from flagella of the temperature-sensitive (ts) flagellar assembly-defective (fla) mutant fla11(ts) at the restrictive temperature. This depletion of CrEB1 is accompanied by a dramatic accumulation of IFT particle polypeptides near the flagellar tip.