Centriolar satellite– and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly

Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction–induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring.     

The Msd1–Wdr8–Pkl1 complex anchors microtubule minus ends to fission yeast spindle pole bodies

The minus ends of spindle microtubules are anchored to a microtubule-organizing center. The conserved Msd1/SSX2IP proteins are localized to the spindle pole body (SPB) and the centrosome in fission yeast and humans, respectively, and play a critical role in microtubule anchoring. In this paper, we show that fission yeast Msd1 forms a ternary complex with another conserved protein, Wdr8, and the minus end–directed Pkl1/kinesin-14. Individual deletion mutants displayed the identical spindle-protrusion phenotypes. Msd1 and Wdr8 were delivered by Pkl1 to mitotic SPBs, where Pkl1 was tethered through Msd1–Wdr8. The spindle-anchoring defect imposed by msd1/wdr8/pkl1 deletions was suppressed by a mutation of the plus end–directed Cut7/kinesin-5, which was shown to be mutual. Intriguingly, Pkl1 motor activity was not required for its anchoring role once targeted to the SPB. Therefore, spindle anchoring through Msd1–Wdr8–Pkl1 is crucial for balancing the Cut7/kinesin-5–mediated outward force at the SPB. Our analysis provides mechanistic insight into the spatiotemporal regulation of two opposing kinesins to ensure mitotic spindle bipolarity.     

Gamma-tubulin complex-mediated anchoring of spindle microtubules to spindle-pole bodies requires Msd1 in fission yeast

The anchoring of microtubules to subcellular structures is critical for cell polarity and motility. Although the process of anchoring cytoplasmic microtubules to the centrosome has been studied in some detail, it is not known how spindle microtubules are anchored to the mitotic centrosome and, particularly, whether anchoring and nucleation of mitotic spindles are functionally separate. Here, we show that a fission yeast coiled-coil protein, Msd1, is required for anchoring the minus end of spindle microtubules to the centrosome equivalent, the spindle-pole body (SPB). msd1 deletion causes spindle microtubules to abnormally extend beyond SPBs, which results in chromosome missegregation. Importantly, this protruding spindle is phenocopied by the amino-terminal deletion mutant of Alp4, a component of the gamma-tubulin complex (gamma-TuC), which lacks the potential Msd1-interacting domain. We propose that Msd1 interacts with gamma-TuC, thereby specifically anchoring the minus end of microtubules to SPBs without affecting microtubule nucleation.     

OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP‐mediated microtubule anchoring

Lysosomal positioning and mTOR (mammalian target of rapamycin) signaling coordinate cellular responses to nutrient levels. Inadequate nutrient sensing can result in growth delays, a hallmark of Lowe syndrome. OCRL mutations cause Lowe syndrome, but the role of OCRL in nutrient sensing is unknown. Here, we show that OCRL is localized to the centrosome by its ASH domain and that it recruits microtubule‐anchoring factor SSX2IP to the centrosome, which is important in the formation of the microtubule‐organizing center. Deficiency of OCRL in human and mouse cells results in loss of microtubule‐organizing centers and impaired microtubule‐based lysosome movement, which in turn leads to mTORC1 inactivation and abnormal nutrient sensing. Centrosome‐targeted PACT‐SSX2IP can restore microtubule anchoring and mTOR activity. Importantly, boosting the activity of mTORC1 restores the nutrient sensing ability of Lowe patients’ cells. Our findings highlight mTORC1 as a novel therapeutic target for Lowe syndrome.     

Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome

Members of the transforming acidic coiled coil (TACC) protein family are emerging as important mitotic spindle assembly proteins in a variety of organisms. The molecular details of how TACC proteins function are unknown, but TACC proteins have been proposed to recruit microtubule-stabilizing proteins of the tumor overexpressed gene (TOG) family to the centrosome and to facilitate their loading onto newly emerging microtubules. Using Xenopus egg extracts and in vitro assays, we show that the Xenopus TACC protein maskin is required for centrosome function beyond recruiting the Xenopus TOG protein XMAP215. The conserved C-terminal TACC domain of maskin is both necessary and sufficient to restore centrosome function in maskin-depleted extracts, and we provide evidence that the N terminus of maskin inhibits the function of the TACC domain. Time-lapse video microscopy reveals that microtubule dynamics in Xenopus egg extracts are unaffected by maskin depletion. Our results provide direct experimental evidence of a role for maskin in centrosome function and suggest that maskin is required for microtubule anchoring at the centrosome.     

The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.     

Microtubule release and capture in epithelial cells.

Many differentiated cells including polarised epithelial cells display a non-radial, apico-basal microtubule array. In some cells the centrosome disassembles and new nucleating sites are created at more appropriate locations. In others the centrosome remains, but relatively few microtubules radiate from it's immediate environs. Instead, the majority of the microtubule minus-ends are associated with apical cell surface sites. Centrosomal microtubule release and capture is evidently a mechanism exploited by some polarised epithelial cells as a means of producing non-centrosomal, apico-basal microtubule arrays. This involves microtubule nucleation at the centrosome, release and subsequent translocation and capture at the apical sites. Two functionally distinct centrosomal complexes dedicated to the control of microtubule nucleation and anchorage have been suggested to be essential and universal features of all centrosomes. The centrosomal proteins ninein and R2 are potential microtubule anchoring proteins and their discovery has exciting implications for centrosomal organisation and microtubule positioning in cells.     

GSK-3beta-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome

Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3beta (GSK-3beta) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3beta is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3beta or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3beta knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3beta and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization.     

The many faces of the bouquet centrosome MTOC in meiosis and germ cell development

Meiotic chromosomal pairing is facilitated by a conserved cytoskeletal organization. Telomeres associate with perinuclear microtubules via Sun/KASH complexes on the nuclear envelope (NE) and dynein. Telomere sliding on perinuclear microtubules contributes to chromosome homology searches and is essential for meiosis. Telomeres ultimately cluster on the NE, facing the centrosome, in a configuration called the chromosomal bouquet. Here, we discuss novel components and functions of the bouquet microtubule organizing center (MTOC) in meiosis, but also broadly in gamete development. The cellular mechanics of chromosome movements and the bouquet MTOC dynamics are striking. The newly identified zygotene cilium mechanically anchors the bouquet centrosome and completes the bouquet MTOC machinery in zebrafish and mice. We hypothesize that various centrosome anchoring strategies evolved in different species. Evidence suggests that the bouquet MTOC machinery is a cellular organizer, linking meiotic mechanisms with gamete development and morphogenesis. We highlight this cytoskeletal organization as a new platform for creating a holistic understanding of early gametogenesis, with direct implications to fertility and reproduction.     

Centrosomal nucleolin is required for microtubule network organization

Nucleolin is a pleiotropic protein involved in a variety of cellular processes. Although multipolar spindle formation has been observed after nucleolin depletion, the roles of nucleolin in centrosome regulation and functions have not been addressed. Here we report using immunofluorescence and biochemically purified centrosomes that nucleolin co-localized only with one of the centrioles during interphase which was further identified as the mature centriole. Upon nucleolin depletion, cells exhibited an amplification of immature centriole markers surrounded by irregular pericentrin staining; these structures were exempt from maturation markers and unable to nucleate microtubules. Furthermore, the microtubule network was disorganized in these cells, exhibiting frequent non-centrosomal microtubules. At the mature centriole a reduced kinetics in the centrosomal microtubule nucleation phase was observed in live silenced cells, as well as a perturbation of microtubule anchoring. Immunoprecipitation experiments showed that nucleolin belongs to protein complexes containing 2 key centrosomal proteins, γ-tubulin and ninein, involved in microtubule nucleation and anchoring steps. Altogether, our study uncovered a new role for nucleolin in restricting microtubule nucleation and anchoring at centrosomes in interphase cells.     

How do Plants Organize Microtubules Without a Centrosome？

A microtubule nucleates from a γ-tubuUn complex, which consists of γ-tubulin, proteins from the SPC971SPC98 family, and the WD40 motif protein GCP-WD. We analyzed the phylogenetic relationships of the genes encoding these proteins and found that the components of this complex are widely conserved among land plants and other eukaryotes. By contrast, the interphase and mitotic arrays of microtubules in land plants differ from those in other eukaryotes. In the interphase cortical array, the majority of microtubules nucleate on existing microtubules in the absence of conspicuous microtubule organizing centers （MTOCs）, such as a centrosome. During mitosis, the spindle also forms in the absence of conspicuous MTOCs. Both poles of the spindle are broad, and branched structures of microtubules called microtubule converging centers form at the poles. In this review, we hypothesize that the microtubule converging centers form via microtubule-dependent microtubule nucleation, as in the case of the interphase arrays. The evolutionary insights arising from the molecular basis of the diversity in microtubule organization are discussed.     

Cep72 regulates the localization of key centrosomal proteins and proper bipolar spindle formation

Microtubule‐nucleation activity and structural integrity of the centrosome are critical for various cellular functions. The γ‐tubulin ring complexes (γTuRCs) localizing to the pericentriolar matrix (PCM) of the centrosome are major sites of microtubule nucleation. The PCM is thought to be created by two cognate large coiled‐coil proteins, pericentrin/kendrin and CG‐NAP/AKAP450, and its stabilization by Kizuna is essential for bipolar spindle formation. However, the mechanisms by which these proteins are recruited and organized into a proper structure with microtubule‐organizing activity are poorly understood. Here we identify a centrosomal protein Cep72 as a Kizuna‐interacting protein. Interestingly, Cep72 is essential for the localization of CG‐NAP and Kizuna. Cep72 is also involved in γTuRC recruitment to the centrosome and CG‐NAP confers the microtubule‐nucleation activity on the γTuRCs. During mitosis, Cep72‐mediated microtubule organization is important for converging spindle microtubules to the centrosomes, which is needed for chromosome alignment and tension generation between kinetochores. Our findings show that Cep72 is the key protein essential for maintaining microtubule‐organizing activity and structural integrity of the centrosome.     

GRASP65 controls Golgi position and structure during G2/M transition by regulating the stability of microtubules

The mammalian Golgi apparatus is organized in the form of a ribbon‐like structure positioned near the centrosome. Despite its multimodular organization, the Golgi complex is characterized by a prominent structural plasticity, which is crucial during essential physiological processes, such as the G2 phase of the cell cycle, during which the Golgi ribbon must be “unlinked” into isolated stacks to allow progression into mitosis. Here we show that the Golgi‐associated protein GRASP65, which is well known for its role in Golgi stacking and ribbon formation, is also required for the organization of the microtubule cytoskeleton. GRASP65 is not involved in microtubule nucleation or anchoring. Instead, it is required for the stabilization of newly nucleated microtubules, leading to their acetylation and clustering of Golgi stacks. Ribbon formation and microtubule stabilization are both regulated by JNK/ERK‐mediated phosphorylation of S274 of GRASP65, suggesting that this protein can coordinate the Golgi structure with microtubule organization. In agreement with an important role, tubulin acetylation is strongly reduced during the G2 phase of the cell cycle, allowing the separation of the Golgi stacks. Thus, our data reveal a fundamental role of GRASP65 in the integration of different stimuli to modulate Golgi structure and microtubule organization during cell division.     

Centrosome control of the cell cycle

Early observations of centrosomes, made a century ago, revealed a tiny dark structure surrounded by a radial array of cytoplasmic fibers. We now know that the fibers are microtubules and that the dark organelles are centrosomes that mediate functions far beyond the more conventional role of microtubule organization. More recent evidence demonstrates that the centrosome serves as a scaffold for anchoring an extensive number of regulatory proteins. Among these are cell-cycle regulators whose association with the centrosome is an essential step in cell-cycle control. Such studies show that the centrosome is required for several cell-cycle transitions, including G(1) to S-phase, G(2) to mitosis and metaphase to anaphase. In this review (which is part of the Chromosome Segregation and Aneuploidy series), we discuss recent data that provide the most direct links between centrosomes and cell-cycle progression.     

Identification of a novel microtubule-destabilizing motif in CPAP that binds to tubulin heterodimers and inhibits microtubule assembly

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the gamma-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.     

Desmoplakin: an unexpected regulator of microtubule organization in the epidermis

Despite their importance in cell shape and polarity generation, the organization of microtubules in differentiated cells and tissues remains relatively unexplored in mammals. We generated transgenic mice in which the epidermis expresses a fluorescently labeled microtubule-binding protein and show that in epidermis and in cultured keratinocytes, microtubules stereotypically reorganize as they differentiate. In basal cells, microtubules form a cytoplasmic network emanating from an apical centrosome. In suprabasal cells, microtubules concentrate at cell-cell junctions. The centrosome retains its ability to nucleate microtubules in differentiated cells, but no longer anchors them. During epidermal differentiation, ninein, which is a centrosomal protein required for microtubule anchoring (Dammermann, A., and A. Merdes. 2002. J. Cell Biol. 159:255-266; Delgehyr, N., J. Sillibourne, and M. Bornens. 2005. J. Cell Sci. 118:1565-1575; Mogensen, M.M., A. Malik, M. Piel, V. Bouckson-Castaing, and M. Bornens. 2000. J. Cell Sci. 113:3013-3023), is lost from the centrosome and is recruited to desmosomes by desmoplakin (DP). Loss of DP prevents accumulation of cortical microtubules in vivo and in vitro. Our work uncovers a differentiation-specific rearrangement of the microtubule cytoskeleton in epidermis, and defines an essential role for DP in the process.     

Dlg1 binds GKAP to control dynein association with microtubules, centrosome positioning, and cell polarity

Centrosome positioning is crucial during cell division, cell differentiation, and for a wide range of cell-polarized functions including migration. In multicellular organisms, centrosome movement across the cytoplasm is thought to result from a balance of forces exerted by the microtubule-associated motor dynein. However, the mechanisms regulating dynein-mediated forces are still unknown. We show here that during wound-induced cell migration, the small G protein Cdc42 acts through the polarity protein Dlg1 to regulate the interaction of dynein with microtubules of the cell front. Dlg1 interacts with dynein via the scaffolding protein GKAP and together, Dlg1, GKAP, and dynein control microtubule dynamics and organization near the cell cortex and promote centrosome positioning. Our results suggest that, by modulating dynein interaction with leading edge microtubules, the evolutionary conserved proteins Dlg1 and GKAP control the forces operating on microtubules and play a fundamental role in centrosome positioning and cell polarity.     

The centrosome keeps nucleating microtubules but looses the ability to anchor them after the inhibition of dynein-dynactin complex

We inhibited dynein in cells either by the expression of coiled coil-1 (CC1) fragment of dynactin p150Glued subunit or by the microinjection of CC1 protein synthesized in Escherichia coli. CC1 impeded the aggregation of pigment granules in fish melanophores and caused the dispersion of Golgi in Vero and HeLa cells. These data demonstrated the inhibiting effect of CC1 on dynein. In cultured cells, CC1 expression caused the disruption of microtubule array, while the nucleation of new microtubules remained unaltered. This was proved both with in vivo microtubule recovery after nocodazole treatment and with in vitro microtubule polymerization on centrosomes, when the number of nucleated microtubules marginally reduced after the incubation with CC1. Moreover, the inhibiting anti-dynein 74.1 antibodies caused the same effect. Thus we have shown that though dynein is not important for microtubule nucleation, it is essential for the radial organization of microtubules presumably being involved in microtubule anchoring on the centrosome. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 11, pp. 1515–1524.     

Centrosome composition and microtubule anchoring mechanisms.

Centrosomes of animal cells and spindle pole bodies of fungi are the major microtubule nucleating centers. Recent studies indicate that their capacity to organize microtubule arrays rests on elaborate control of the anchoring and release of the nucleated microtubules. Although common molecular mechanisms are likely to be involved in both cases, the centrosome from animal cells shows considerable complexity and flexibility, which contrasts with the simple laminar organization of spindle pole bodies in fungi. The role of the centriole pair in controlling both the structural stability and the activity of the centrosome in animal cells is now becoming clearer. The potential use of the generational asymmetry of centrosomes or spindle pole bodies for controlling cell polarity is also a growing theme.     

Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans Embryos

gamma-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.