Centriolar Microtubules Research Articles

Centriolar Kinesin Kif24 Interacts with CP110 to Remodel Microtubules and Regulate Ciliogenesis

We have identified a protein, Kif24, that shares homology with the kinesin-13 subfamily of motor proteins and specifically interacts with CP110 and Cep97, centrosomal proteins that play a role in regulating centriolar length and ciliogenesis. Kif24 preferentially localizes to mother centrioles. Loss of Kif24 from cycling cells resulted in aberrant cilia assembly but did not promote growth of abnormally long centrioles, unlike CP110 and Cep97 depletion. We found that loss of Kif24 leads to the disappearance of CP110 from mother centrioles, specifically in cycling cells able to form cilia. Kif24 is able to bind and depolymerize microtubules in vitro. Remarkably, ectopically expressed Kif24 specifically remodels centriolar microtubules without significantly altering cytoplasmic microtubules. Thus, our studies have identified a centriolar kinesin that specifically remodels a subset of microtubules, thereby regulating cilia assembly. These studies also suggest mechanistic differences between the regulation of microtubule elongation associated with centrioles and cilia.

Ofd1, a Human Disease Gene, Regulates the Length and Distal Structure of Centrioles

Centrosomes and their component centrioles represent the principal microtubule organizing centers of animal cells. Here, we show that the gene underlying orofaciodigital syndrome 1, Ofd1, is a component of the distal centriole that controls centriole length. In the absence of Ofd1, distal regions of centrioles, but not procentrioles, elongate abnormally. These long centrioles are structurally similar to normal centrioles but contain destabilized microtubules with abnormal posttranslational modifications. Ofd1 is also important for centriole distal appendage formation and centriolar recruitment of the intraflagellar transport protein Ift88. To model OFD1 syndrome in embryonic stem cells, we replaced the Ofd1 gene with missense alleles from human OFD1 patients. Distinct disease-associated mutations cause different degrees of excessive or decreased centriole elongation, all of which are associated with diminished ciliogenesis. Our results indicate that Ofd1 acts at the distal centriole to build distal appendages, recruit Ift88, and stabilize centriolar microtubules at a defined length.

Self-assembling SAS-6 Multimer Is a Core Centriole Building Block

Centrioles are conserved microtubule-based organelles with 9-fold symmetry that are essential for cilia and mitotic spindle formation. A conserved structure at the onset of centriole assembly is a "cartwheel" with 9-fold radial symmetry and a central tubule in its core. It remains unclear how the cartwheel is formed. The conserved centriole protein, SAS-6, is a cartwheel component that functions early in centriole formation. Here, combining biochemistry and electron microscopy, we characterize SAS-6 and show that it self-assembles into stable tetramers, which serve as building blocks for the central tubule. These results suggest that SAS-6 self-assembly may be an initial step in the formation of the cartwheel that provides the 9-fold symmetry. Electron microscopy of centrosomes identified 25-nm central tubules with repeating subunits and show that SAS-6 concentrates at the core of the cartwheel. Recombinant and native SAS-6 self-oligomerizes into tetramers with approximately 6-nm subunits, and these tetramers are components of the centrosome, suggesting that tetramers are the building blocks of the central tubule. This is further supported by the observation that elevated levels of SAS-6 in Drosophila cells resulted in higher order structures resembling central tubule morphology. Finally, in the presence of embryonic extract, SAS-6 tetramers assembled into high density complexes, providing a starting point for the eventual in vitro reconstruction of centrioles.

γ-Tubulin-containing abnormal centrioles are induced by insufficient Plk4 in human HCT116 colorectal cancer cells

Cancer cells frequently induce aberrant centrosomes, which have been implicated in cancer initiation and progression. Human colorectal cancer cells, HCT116, contain aberrant centrioles composed of disorganized cylindrical microtubules and displaced appendages. These cells also express unique centrosome-related structures associated with a subset of centrosomal components, including gamma-tubulin, centrin and PCM1. During hydroxyurea treatment, these abnormal structures become more abundant and undergo a change in shape from small dots to elongated fibers. Although gamma-tubulin seems to exist as a ring complex, the abnormal structures do not support microtubule nucleation. Several lines of evidence suggest that the fibers correspond to a disorganized form of centriolar microtubules. Plk4, a mammalian homolog of ZYG-1 essential for initiation of centriole biogenesis, is not associated with the gamma-tubulin-specific abnormal centrosomes. The amount of Plk4 at each centrosome was less in cells with abnormal centrosomes than cells without gamma-tubulin-specific abnormal centrosomes. In addition, the formation of abnormal structures was abolished by expression of exogenous Plk4, but not SAS6 and Cep135/Bld10p, which are downstream regulators required for the organization of nine-triplet microtubules. These results suggest that HCT116 cells fail to organize the ninefold symmetry of centrioles due to insufficient Plk4.

Cellular footprints of plant evolution—news from the blepharoblast

The microtubular cytoskeleton of higher plants diverges considerably from its animal counterpart. This divergence involves a fundamentally different organization with microtubule arrays, which are specific to higher plants, such as cortical microtubules or the phragmoplast. On the other hand, centrioles, which are central organizers of microtubules in cells of animals and lower plants, have been progressively reduced in the course of plant evolution, and are now absent, as in most seed plants. The role of centrioles as major microtubule-organizing centers (MTOC) apparently has been taken over by the nuclear envelope. However, the spermatogenous cells of pteridophytes and some of the primitive gymnosperms are still endowed with centriolar structures, which is probably related to the fact that the spermatozoids move by flagella. In other words: these specialized cells represent something like a “missing link” between the ancestral situation, where microtubules are nucleated from persistent microtubule rods that are organized in a specialized organelle, and a derived situation, where microtubules emerge from more or less diffuse MTOCs. In the pteridophytes, the centriolar structure is known as the blepharoplast and arises in the last two division cycles. It organizes the interphase array of microtubules along with the spindle for the two final divisions. Prior to each of these mitotic events, the blepharoplast divides itself and moves to the poles, serving as the focus for an astral spindle. After the final division, it re-organizes to a much looser structure and basal bodies are produced that will eventually organize the sperm flagellar apparatus. The work by Vaughn and Bowling in the present issue highlights this evolutionarily interesting transition. Using spermatogenous cells of the fern Ceratopteris richardii, they investigate the effect of transient elimination of microtubules. They use oryzalin, an inhibitor that sequesters tubulin heterodimers from being incorporated into the growing microtubule, such that microtubules shorten and eventually disappear as consequence of their innate turnover. The blepharoplast contains barrel-like structures that serve as centriolar templates embedded in an electronopaque pericentriolar matrix. Upon treatment with oryzalin they observe that this pericentriolar material forms clear gaps that correspond in their dimension to the typical microtubular diameter and stain with γ-tubulin antibodies. Despite the absence of microtubules, the division of the blepharoblast is observed to continue—this means that the division of the MTOC seems to be independent of the presence of microtubules. This represents an evolutionary “modern” trait heralding the situation in higher plant cells, where microtubule arrays cycle although there are no centriolar microtubule asters. The gaps might be morphological manifestations of the γ-tubulin ring complexes that nucleate microtubules in different phyla. After washout, the microtubule arrays showed malformations or multiplications that mirrored the behaviour of the blepharoblasts during drug treatment. Therefore, in terms of cell biology, the spermatogenous cells combines an evolutionarily ancient situation, namely, dependence of microtubule organization from a centriolar organelle, with an evolutionarily derived trait, independence of microtubule organizers from preexisting microtubules. These cells are therefore exciting models to follow the cellular footprints of plant evolution. Protoplasma (2008) 233:175 DOI 10.1007/s00709-008-0009-4

SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the γ-tubulin–mediated addition of centriolar microtubules

Centrioles are surrounded by pericentriolar material (PCM), which is proposed to promote new centriole assembly by concentrating γ-tubulin. Here, we quantitatively monitor new centriole assembly in living Caenorhabditis elegans embryos, focusing on the conserved components SAS-4 and SAS-6. We show that SAS-4 and SAS-6 are coordinately recruited to the site of new centriole assembly and reach their maximum levels during S phase. Centriolar SAS-6 is subsequently reduced by a mechanism intrinsic to the early assembly pathway that does not require progression into mitosis. Centriolar SAS-4 remains in dynamic equilibrium with the cytoplasmic pool until late prophase, when it is stably incorporated in a step that requires γ-tubulin and microtubule assembly. These results indicate that γ-tubulin in the PCM stabilizes the nascent daughter centriole by promoting microtubule addition to its outer wall. Such a mechanism may help restrict new centriole assembly to the vicinity of preexisting parent centrioles that recruit PCM.

DSAS-6 Organizes a Tube-like Centriole Precursor, and Its Absence Suggests Modularity in Centriole Assembly

Centrioles are microtubule-based cylindrical structures that exhibit 9-fold symmetry and facilitate the organization of centrosomes, flagella, and cilia [1]. Abnormalities in centrosome structure and number occur in many cancers [1, 2]. Despite its importance, very little is known about centriole biogenesis. Recent studies in C. elegans have highlighted a group of molecules necessary for centriole assembly [1, 3]. ZYG-1 kinase recruits a complex of two coiled-coil proteins, SAS-6 and SAS-5, which are necessary to form the C. elegans centriolar tube, a scaffold in centriole formation [4, 5]. This complex also recruits SAS-4, which is required for the assembly of the centriolar microtubules that decorate that tube [4, 5]. Here we show that Drosophila SAS-6 is involved in centriole assembly and cohesion. Overexpression of DSAS-6 in syncitial embryos led to the de novo formation of multiple microtubule-organizing centers (MTOCs). Strikingly, the center of these MTOCs did not contain centrioles, as described previously for SAK/PLK4 overexpression [6]. Instead, tube-like structures were present, supporting the idea that centriolar assembly starts with the formation of a tube-like scaffold, dependent on DSAS-6 [5]. In DSAS-6 loss-of-function mutants, centrioles failed to close and to elongate the structure along all axes of the 9-fold symmetry, suggesting modularity in centriole assembly. We propose that the tube is built from nine subunits fitting together laterally and longitudinally in a modular and sequential fashion, like pieces of a layered "hollow" cake.

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.

Plant microtubule studies: past and present

Here, I briefly review historical and morphological aspects of plant microtubule studies in land plants. Microtubules are formed from tubulins, and the polymeric configurations appear as singlet, doublet, and triplet microtubules. Doublet microtubules occur in the axoneme of cilia and flagella, and triplet microtubules occur in the basal bodies and centrosomes. Doublet and triplet microtubules are lost in all angiosperms and some gymnosperms that do not possess flagellated sperm. In land plants with flagellated sperm, centriolar centrosomes transform into basal bodies during spermatogenesis. In flowering plants, however, most male gametes (sperm) are conveyed to eggs without the benefit of cilia or flagella; thus, higher plants lack centriolar centrosome and doublet and triplet microtubules. The loss of centriolar centrosomes from the life cycle of flowering plants may have influenced the evolution of the plant microtubule system. Comparison of mitotic apparatuses in basal land plants and flowering plants illuminates the evolutionary transition from the centriolar microtubule system to the acentriolar microtubule system.

Making Microtubules and Mitotic Spindles in Cells without Functional Centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.

Elongation of centriolar microtubule triplets contributes to the formation of the mitotic spindle in gamma-tubulin-depleted cells.

The assembly of the mitotic spindle after depletion of the major gamma-tubulin isotype by RNA-mediated interference was assessed in the Drosophila S2 cell line. Depletion of gamma-tubulin had no significant effect on the cytoskeletal microtubules during interphase. However, it promoted an increase in the mitotic index, resulting mainly in monopolar and, to a lesser extent, asymmetrical bipolar prometaphases lacking astral microtubules. This mitotic accumulation coincided with the activation of the mitotic checkpoint. Immunostaining with an anti-Asp antibody revealed that the spindle poles, which were always devoid of gamma-tubulin, were unfocused and organized into sub-spindles. Despite the marked depletion of gamma-tubulin, the pericentriolar proteins CP190 and centrosomin were recruited to the spindle pole(s), where they formed three or four dots, suggesting the presence of several centrioles. Electron microscopic reconstructions demonstrated that most of the monopolar spindles exhibited three or four centrioles, indicating centriole duplication with a failure in the separation process. Most of the centrioles were shortened, suggesting a role for gamma-tubulin in centriole morphogenesis. Moreover, in contrast to metaphases observed in control cells, in which the spindle microtubules radiated from the pericentriolar material, in gamma-tubulin-depleted cells, microtubule assembly still occurred at the poles but involved the elongation of centriolar microtubule triplets. Our results demonstrate that, after depletion of gamma-tubulin, the pericentriolar material is unable to promote efficient microtubule nucleation. They point to an alternative mechanism of centrosomal microtubule assembly that contributes to the formation of abnormal, albeit partially functional, mitotic spindles.

Functional role of epsilon-tubulin in the assembly of the centriolar microtubule scaffold.

Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as δ-, ɛ-, or η-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of ɛ-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules.

Centrosome reduction during rhesus spermiogenesis: γ‐tubulin, centrin, and centriole degeneration

Centrosome reduction during spermiogenesis has been studied using anti-γ-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with γ-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard γ-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable γ-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice. Mol. Reprod. Dev. 56:502–511, 2000. © 2000 Wiley-Liss, Inc.

Highly degenerated distal centrioles in rhesus and human spermatozoa.

In humans and other mammals except rodents, the spermatozoa contribute the proximal centriole during fertilization. The inheritance of the distal centriole is not yet fully clear. In the present work, the distal centrioles of rhesus and human spermatozoa have been studied by transmission electron microscopy. The round and elongating rhesus spermatids possess both proximal and distal centrioles. The distal centriole extends posteriorly as an axoneme while the proximal centriole produces a microtubular adjunct. Ejaculated rhesus and human spermatozoa have intact proximal centrioles, but the distal centrioles have degenerated. The central pair of microtubules of the axoneme extends continuously into the distal centriolar region up to the sperm head. Serial transverse and longitudinal sections of the sperm neck region reveal few scattered microtubule duplexes or triplets in the distal centriolar region. The loss of the centriolar microtubules is more extensive on the ventral side of the neck region, the side where the proximal centriole resides. The distal centriole degenerates caudally from the rostral area. Immunogold electron microscopy with anti-beta-tubulin antibody showed that the distal centriolar regions possess 50% fewer gold particles than the proximal centrioles, indicating a significant loss of centriolar microtubules in the distal centriolar region. The A-tubules of the remaining triplets are filled with a dense material, as observed in the axoneme. Thus, rhesus and human spermatozoa introduce only proximal centrioles intact, whereas the distal centrioles are mostly disorganized in the mature spermatozoa.

Centrosome reduction during Rhesus spermiogenesis: gamma-tubulin, centrin, and centriole degeneration.

Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.

The centrosome

The centrosome

Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells.

Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223-232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population. During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis. Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.

The core of the mammalian centriole contains gamma-tubulin.

The microtubule network, upon which transport occurs in higher cells, is formed by the polymerization of alpha and beta tubulin. The third major tubulin isoform, gamma tubulin, is believed to serve a role in organizing this network by nucleating microtubule growth on microtubule-organizing centers, such as the centrosome. Research in vitro has shown that gamma tubulin must be restored to stripped centrioles to regenerate the centrosomal functions of duplication and microtubule nucleation. We have re-examined the localization of gamma tubulin in isolated and in situ mammalian centrosomes using a novel immunocytochemical technique that preserves antigenicity and morphology while allowing increased accessibility. As expected, alpha tubulin was localized in cytoplasmic and centriolar barrel microtubules and in the associated pericentriolar material. Foci of gamma tubulin were observed at the periphery of the organized pericentriolar material, as reported previously, often near the termini of microtubules. A further and major location of gamma tubulin was a structure within the proximal end of the centriolar barrel. The distributions were complementary, in that alpha tubulin was excluded from the core of the centriole, and gamma tubulin was excluded from the microtubule barrel. We have shown that gamma tubulin is localized both in the pericentriolar material and in the core of the mammalian centriole. This result suggests that gamma tubulin has a role in the centriolar duplication process, perhaps as a template for growth of the centriolar microtubules, in addition to its established role in the nucleation of astral microtubules.

Spermatozoa of megaloptera and raphidioptera (insecta, neuropteroidea)

Spermatozoa from representatives of two minor insect orders, Sialis lutaria, order Megaloptera, and Raphidia spec., order Raphidioptera, have been examined by electron microscopy. Spermatozoa from both species were seen to have an elongated sperm head and a sperm tail containing axoneme, two mitochondrial derivatives of equal sizes, and two accessory bodies. The acrosome in Sialis has a bilateral symmetry in cross section and is fairly complex in that it has both a thin anterior extra-acrosomal layer, an acrosomal vesicle, and a subacrosomal space. In the acrosomal vesicle there is a filament bundle that makes a U-turn. The centriolar microtubules fray out to surround an attenuated posterior portion of the nucleus. The spermatozoa of Raphidia are very long and thin and have a rather simple acrosome. In this species, too, the microtubules of the centrioles surround the posterior, 1-μm-long, portion of the nucleus. This configuration of the centriolar microtubules is not shared by the other examined insects of the neuropteroid superorder and may be a synapomorphic feature. The data are thus consistent with the opinion that Megaloptera plus Raphidioptera form a sister group to the Neuroptera (Planipennia).

A gene encoding the major beta tubulin of the mitotic spindle in Physarum polycephalum plasmodia.

The multinucleate plasmodium of Physarum polycephalum is unusual among eucaryotic cells in that it uses tubulins only in mitotic-spindle microtubules; cytoskeletal, flagellar, and centriolar microtubules are absent in this cell type. We have identified a beta-tubulin cDNA clone, beta 105, which is shown to correspond to the transcript of the betC beta-tubulin locus and to encode beta 2 tubulin, the beta tubulin expressed specifically in the plasmodium and used exclusively in the mitotic spindle. Physarum amoebae utilize tubulins in the cytoskeleton, centrioles, and flagella, in addition to the mitotic spindle. Sequence analysis shows that beta 2 tubulin is only 83% identical to the two beta tubulins expressed in amoebae. This compares with 70 to 83% identity between Physarum beta 2 tubulin and the beta tubulins of yeasts, fungi, alga, trypanosome, fruit fly, chicken, and mouse. On the other hand, Physarum beta 2 tubulin is no more similar to, for example, Aspergillus beta tubulins than it is to those of Drosophila melanogaster or mammals. Several eucaryotes express at least one widely diverged beta tubulin as well as one or more beta tubulins that conform more closely to a consensus beta-tubulin sequence. We suggest that beta-tubulins diverge more when their expression pattern is restricted, especially when this restriction results in their use in fewer functions. This divergence among beta tubulins could have resulted through neutral drift. For example, exclusive use of Physarum beta 2 tubulin in the spindle may have allowed more amino acid substitutions than would be functionally tolerable in the beta tubulins that are utilized in multiple microtubular organelles. Alternatively, restricted use of beta tubulins may allow positive selection to operate more freely to refine beta-tubulin function.