The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation

Cilia and basal bodies are essential organelles for a broad spectrum of functions, including the development of left-right asymmetry, kidney function, cerebrospinal fluid transport, generation of photoreceptor outer segments, and hedgehog signaling. Zebrafish fleer (flr) mutants exhibit kidney cysts, randomized left-right asymmetry, hydrocephalus, and rod outer segment defects, suggesting a pleiotropic defect in ciliogenesis. Positional cloning flr identified a tetratricopeptide repeat protein homologous to the Caenorhabditis elegans protein DYF1 that was highly expressed in ciliated cells. flr pronephric cilia were shortened and showed a reduced beat amplitude, and olfactory cilia were absent in mutants. flr cilia exhibited ultrastructural defects in microtubule B-tubules, similar to axonemes that lack tubulin posttranslational modifications (polyglutamylation or polyglycylation). flr cilia showed a dramatic reduction in cilia polyglutamylated tubulin, indicating that flr encodes a novel modulator of tubulin polyglutamylation. We also found that the C. elegans flr homologue, dyf-1, is also required for tubulin polyglutamylation in sensory neuron cilia. Knockdown of zebrafish Ttll6, a tubulin polyglutamylase, specifically eliminated tubulin polyglutamylation and cilia formation in olfactory placodes, similar to flr mutants. These results are the first in vivo evidence that tubulin polyglutamylation is required for vertebrate cilia motility and structure, and, when compromised, results in failed ciliogenesis.     

Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole- associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Replication of basal bodies and centrioles.

During the past year, studies on the centrioles and basal bodies of animal and algal cells, and the spindle pole bodies of yeast and other fungi, have added significantly to our knowledge of how these cell organelles form and how they function in initiating microtubule assembly throughout the cell cycle. Most of these studies have used antibodies to identify proteins within and around these organelles and, in some cases, to disrupt their ability to nucleate microtubules. Genetic methods have been used to identify specific proteins, including a new member of the tubulin superfamily, involved in the function and replication of spindle pole bodies and centrioles.     

The microtubular system and posttranslationally modified tubulin during spermatogenesis in a parasitic nematode with amoeboid and aflagellate spermatozoa

Using transmission electron microscopy and immunologic approaches with various antibodies against general tubulin and posttranslationally modified tubulin, we investigated microtubule organization during spermatogenesis in Heligmosomoides polygyrus, a species in which a conspicuous but transient microtubular system exists in several forms: a cytoplasmic network in the spermatocyte, the meiotic spindle, a perinuclear network and a longitudinal bundle of microtubules in the spermatid. This pattern differs from most nematodes including Caenorhabditis elegans, in which spermatids have not microtubules. In the spermatozoon of H. polygyrus, immunocytochemistry does not detect tubulin, but electron microscopy reveals two centrioles with a unique structure of 10 singlets. In male germ cells, microtubules are probably involved in cell shaping and positioning of organelles but not in cell motility. In all transient tubulin structures described in spermatocytes and spermatids of H. polygyrus, detyrosination, tyrosination, and polyglutamylation were detected, but acetylation and polyglycylation were not. The presence/absence of these posttranslational modifications is apparently not stage dependent. This is the first study of posttranslationally modified tubulin in nematode spermatogenesis. Mol. Reprod. Dev. 49:150–167, 1998. © 1998 Wiley-Liss, Inc.     

Isotype expression, post-translational modification and stage-dependent production of tubulins in erythrocytic Plasmodium falciparum

Microtubules are cytoskeletal polymers containing repeating alpha/beta-tubulin heterodimers and are found in all eukaryotes including the malaria parasite Plasmodium falciparum. Diverse cellular functions such as chromosomal segregation, organelle transport and the determination of cell shape and motility are all dependent on microtubules. This essential role played by tubulin in cells is reflected in the effective use of anti-microtubule agents as fungicides, herbicides, anti-parasitic and anti-cancer agents. Plasmodium falciparum microtubules have been proposed as a potential antimalarial drug target and knowledge of their molecular composition and cellular architecture in blood-stage parasites is required to substantiate this premise. We report here that: (i) the two alpha-tubulin isotypes, alphaI- and alphaII-tubulin, are produced in both asexual and sexual blood-stage parasites, contrary to the previous report that alphaII-tubulin was specific to male gametocytes; (ii) tubulin production is highly stage-dependent in asexual parasites, reaching its maximum level in schizonts and segmenters and (iii) there is evidence of post-translational polyglutamylation of tubulin. The glutamylation of P. falciparum tubulins is the first reported post-translational modification of tubulin in this organism and was found only in the microtubule-organising centres and post-mitotic microtubular structures, suggesting possible roles for this modification in spindle pole body formation and merozoite biogenesis. Taken together, these findings form the basis for a better biological appreciation of P. falciparum microtubules and for the correct deployment of purified tubulins in the evaluation of microtubule inhibitors as potential antimalarial drugs.     

Common themes in centriole and centrosome movements

Centrioles are found in nearly all eukaryotic cells and are required for growth and maintenance of the radial array of microtubules, the mitotic spindle, and cilia and flagella. Different types of microtubule structures are often required at different places in a given cell; centrioles must move around to nucleate these varied structures. Here, we draw together recent data on diverse centriole movements to decipher common themes in how centrioles move. Par proteins establish and maintain the required cellular asymmetry. The actin cytoskeleton facilitates movement of multiple basal bodies. Microtubule forces acting on the cell cortex, and nuclear-cytoskeletal links, are important for positioning individual centrosomes, and during cell division. Knowledge of these common mechanisms can inform the study of centriole movements across biology.     

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.     

Glutamylated tubulin: diversity of expression and distribution of isoforms

Glutamylation of alpha and beta tubulin isotypes is a major posttranslational modification giving rise to diversified isoforms occurring mainly in neurotubules, centrioles, and axonemes. Monoglutamylated tubulin isoforms can be differentially recognized by two mAbs, B3 and GT335, which both recognize either polyglutamylated isoforms. In the present study, immunoelectron microscopy and immunofluorescence analyses were performed with these two mAbs to determine the expression and distribution of glutamylated tubulin isoforms in selected biological models whose tubulin isotypes are characterized. In mouse spermatozoa, microtubules of the flagellum contain polyglutamylated isoforms except in the tip where only monoglutamylated isoforms are detected. In spermatids, only a subset of manchette microtubules contain monoglutamylated tubulin isoforms. Cytoplasmic microtubules of Sertoli cells are monoglutamylated. Mitotic and meiotic spindles of germ cells are monoglutamylated whereas the HeLa cell mitotic spindle is polyglutamylated. Three models of axonemes are demonstrated as a function of the degree and extent of tubulin glutamylation. In lung ciliated cells, axonemes are uniformly polyglutamylated. In sea urchin sperm and Chlamydomonas, flagellar microtubules are polyglutamylated in their proximal part and monoglutamylated in their distal part. In Paramecium, cilia are bi- or monoglutamylated only at their base. In all cells, centrioles or basal bodies are polyglutamylated. These new data emphasize the importance of glutamylation in all types of microtubules and strengthen the hypothesis of its role in the regulation of the intracellular traffic and flagellar motility.     

Polyglutamylated Tubulin Binding Protein C1orf96/CSAP Is Involved in Microtubule Stabilization in Mitotic Spindles

The centrosome-associated C1orf96/Centriole, Cilia and Spindle-Associated Protein (CSAP) targets polyglutamylated tubulin in mitotic microtubules (MTs). Loss of CSAP causes critical defects in brain development; however, it is unclear how CSAP association with MTs affects mitosis progression. In this study, we explored the molecular mechanisms of the interaction of CSAP with mitotic spindles. Loss of CSAP caused MT instability in mitotic spindles and resulted in mislocalization of Nuclear protein that associates with the Mitotic Apparatus (NuMA), with defective MT dynamics. Thus, CSAP overload in the spindles caused extensive MT stabilization and recruitment of NuMA. Moreover, MT stabilization by CSAP led to high levels of polyglutamylation on MTs. MT depolymerization by cold or nocodazole treatment was inhibited by CSAP binding. Live-cell imaging analysis suggested that CSAP-dependent MT-stabilization led to centrosome-free MT aster formation immediately upon nuclear envelope breakdown without γ-tubulin. We therefore propose that CSAP associates with MTs around centrosomes to stabilize MTs during mitosis, ensuring proper bipolar spindle formation and maintenance.     

Taxol-requiring mutant of Chinese hamster ovary cells with impaired mitotic spindle assembly

In the accompanying paper (Cabral, F., 1982, J. Cell. Biol., 97:22-29) we described the isolation and properties of taxol-requiring mutants of Chinese hamster ovary cells. We now show that at least one of these mutants, Tax-18, has an impaired ability to form a spindle apparatus. Immunofluorescence studies using antibodies to tubulin demonstrate that, when incubated in the absence of taxol, Tax-18 forms only a rudimentary spindle with few and shortened microtubules associated with the spindle poles. Furthermore, midbodies were not observed, consistent with an absence of cytokinesis. Essentially normal spindles and midbodies are seen in the presence of taxol. Electron microscopic examination indicates that centrioles and kinetochores are morphologically normal in the mutant strain. Pole-to-kinetochore microtubules were seen but interpolar microtubules were not. Taxol-deprived mutant cells stained with anti-centrosome serum show an elevated centriole content, indicating that the defect in Tax-18 does not affect centriole replication or prevent progression through the cell cycle. Although Tax-18 cells do not form a complete spindle in the absence of taxol, cytoplasmic microtubule assembly occurs in association with microtubule-organizing centers, and microtubules with apparently normal morphology exist throughout the cytoplasm. Observation of chromosome movement indicates that the defect in these cells occurs after prometaphase. These studies demonstrate that the formation of spindle microtubules requires cellular conditions that are different from those required for cytoplasmic microtubule formation. They further show that a normal spindle may be necessary for cytokinesis but not for progress of the cells through the cell cycle.     

Localization of tubulin in the mitotic apparatus of mammalian cells by immunofluorescence and immunoelectron microscopy

Antitubulin antibody was used as an immunofluorescent and immunoelectron microscopic probe to localize tubulin in components of the mitotic apparatus of rat kangaroo (strain PtK₁) cells in vitro. In addition to the detection of tubulin in the spindle microtubules and centrioles, other structures were found to display specific staining including kinetochores, amorphous pericentriolar material and small virus-like particles associated with the centrioles. The kinetochores consisted of a densely stained outer layer about 400 Å thick which is separated from an inner layer of the same dimension by a lightly staining middle layer. Microtubules were primarily associated with the outermost plate of the kinetochore but tubulin was uniformly distributed in both outer and inner plates. Colcemid treatment prevented the assembly of spindle microtubules and resulted in specific alterations of the kinetochore but failed to diminish the staining of the kinetochores. These observations suggest that tubulin molecules may comprise an important structural component of the kinetochore.     

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.     

Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.     

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.     

Polyglutamylation of atlantic cod tubulin: immunochemical localization and possible role in pigment granule transport

In higher organisms, there is a large variety of tubulin isoforms, due to multiple tubulin genes and extensive post-translational modification. The properties of microtubules may be modulated by their tubulin isoform composition. Polyglutamylation is a post-translational modification that is thought to influence binding of both structural microtubule associated proteins (MAPs) and mechano-chemical motors to tubulin. The present study investigates the role of tubulin polyglutamylation in a vesicle transporting system, cod (Gadus morhua) melanophores. We did this by microinjecting an antibody against polyglutamylated tubulin into these cells. To put our results into perspective, and to be able to judge their universal application, we characterized cod tubulin polyglutamylation by Western blotting technique, and compared it to what is known from mammals. We found high levels of polyglutamylation in tissues and cell types whose functions are highly dependent on interactions between microtubules and motor proteins. Microinjection of the anti-polyglutamylation antibody GT335 into cultured melanophores interfered with pigment granule dispersion, while dynein-dependent aggregation was unaffected. Additional experiments showed that GT335-injected cells were able to aggregate pigment even when actin filaments were depolymerized, indicating that the maintained ability of pigment aggregation in these cells was indeed microtubule-based and did not depend upon actin filaments. The results indicate that dynein and the kinesin-like dispersing motor protein in cod melanophores bind to tubulin on slightly different sites, and perhaps depend differentially on polyglutamylation for their interaction with microtubules. The binding site of the dispersing motor may bind directly to the polyglutamate chain, or more closely than dynein.     

Consequences of defective tubulin folding on heterodimer levels, mitosis and spindle morphology in Saccharomyces cerevisiae

In budding yeast, the essential roles of microtubules include segregating chromosomes and positioning the nucleus during mitosis. Defects in these functions can lead to aneuploidy and cell death. To ensure proper mitotic spindle and cytoplasmic microtubule formation, the cell must maintain appropriate stoichiometries of alpha- and beta-tubulin, the basic subunits of microtubules. The experiments described here investigate the minimal levels of tubulin heterodimers needed for mitotic function. We have found a triple-mutant strain, pac10Delta plp1Delta yap4Delta, which has only 20% of wild-type tubulin heterodimer levels due to synthesis and folding defects. The anaphase spindles in these cells are approximately 64% the length of wild-type spindles. The mutant cells are viable and accurately segregate chromosomes in mitosis, but they do have specific defects in mitosis such as abnormal nuclear positioning. The results establish that cells with 20% of wild-type levels of tubulin heterodimers can perform essential cellular functions with a short spindle, but require higher tubulin heterodimer concentrations to attain normal spindle length and prevent mitotic defects.     

Tubulin assembly protein: immunochemical and immunofluorescent studies on its function and distribution in microtubules and cultured cells.

Cytoplasmic microtubule assembly from tubulin monomers requires an accessory protein or proteins present is isolated microtubules. These proteins have been designated "tau" factors. One such factor, tubulin assembly protein (TAP), has been purified to homogeneity from calf brain microtubules. A precipitating, monospecific antibody against the protein has been prepared. The antibody has been used to investigate the mechanism of TAP action in microtubule assembly and the distribution of TAP in cellular microtubules. Immunochemical, immunofluorescent and electron microscopic studies indicate that TAP functions stoichiometrically by binding physically to tubulin to form a complex active in microtubule assembly. TAP is an elongation protein which is required throughout the growth of a microtubule and which is actually present along the entire microtubule. Immunofluorescence microscopy has been used to demonstrate that TAP is distributed throughout the cytoplasmic microtubule network of cultured human, hamster and rat cells-both normal and virally transformed. Immunofluorescence of cells in mitosis shows that TAP is present in the mitotic spindle. These results demonstrate the biological importance of tubulin assembly protein and suggest that it or immunologically related "tau" proteins represent ubiquitous cofactors in cytoplasmic microtubule assembly.     

The Tubulin Code

Microtubules create diverse arrays with specific cellular functions such as the mitotic spindle, cilia, and bundles inside neurons. How microtubules are regulated to enable specific functions is not well understood. Recent work has shown that posttranslational modifications of the tubulin building blocks mark subpopulations of microtubules and regulate downstream microtubule-based functions. In this way, the tubulin modifications generate a “code” that can be read by microtubule-associated proteins in a manner analogous to how the histone code directs diverse chromatin functions. Here we review recent progress in understanding how the tubulin code is generated, maintained, and read by microtubule effectors.     

CENP-W Plays a Role in Maintaining Bipolar Spindle Structure

The CENP-W/T complex was previously reported to be required for mitosis. HeLa cells depleted of CENP-W displayed profound mitotic defects, with mitotic timing delay, disorganized prometaphases and multipolar spindles as major phenotypic consequences. In this study, we examined the process of multipolar spindle formation induced by CENP-W depletion. Depletion of CENP-W in HeLa cells labeled with histone H2B and tubulin fluorescent proteins induced rapid fragmentation of originally bipolar spindles in a high proportion of cells. CENP-W depletion was associated with depletion of Hec1 at kinetochores. The possibility of promiscuous centrosomal duplication was ruled out by immunofluorescent examination of centrioles. However, centrioles were frequently observed to be abnormally split. In addition, a large proportion of the supernumerary poles lacked centrioles, but were positively stained with different centrosomal markers. These observations suggested that perturbation in spindle force distribution caused by defective kinetochores could contribute to a mechanical mechanism for spindle pole disruption. ‘Spindle free’ nocodazole arrested cells did not exhibit pole fragmentation after CENP-W depletion, showing that pole fragmentation is microtubule dependent. Inhibition of centrosome separation by monastrol reduced the incidence of spindle pole fragmentation, indicating that Eg5 plays a role in spindle pole disruption. Surprisingly, CENP-W depletion rescued the monopolar spindle phenotype of monastrol treatment, with an increased frequency of bipolar spindles observed after CENP-W RNAi. We overexpressed the microtubule cross-linking protein TPX2 to create spindle poles stabilized by the microtubule cross-linking activity of TPX2. Spindle pole fragmentation was suppressed in a TPX2-dependent fashion. We propose that CENP-W, by influencing proper kinetochore assembly, particularly microtubule docking sites, can confer spindle pole resistance to traction forces exerted by motor proteins during chromosome congression. Taken together, our findings are consistent with a model in which centrosome integrity is controlled by the pathways regulating kinetochore-microtubule attachment stability.