Centrobin regulates centrosome function in interphase cells by limiting pericentriolar matrix recruitment

The amount of pericentriolar matrix at the centrosome is tightly linked to both microtubule nucleation and centriole duplication, although the exact mechanism by which pericentriolar matrix levels are regulated is unclear. Here we show that Centrobin, a centrosomal protein, is involved in regulating these levels. Interphase microtubule arrays in Centrobin-depleted cells are more focused around the centrosome and are less stable than the arrays in control cells. Centrobin-depleted cells initiate microtubule nucleation more rapidly than control cells and exhibit an increase in the number of growing microtubule ends emanating from the centrosome, while the parameters of microtubule plus end dynamics around the centrosome are not significantly altered. Finally, we show that Centrobin depletion results in the increased recruitment of pericentriolar matrix proteins to the centrosome, including γ-tubulin, AKAP450, Kendrin and PCM-1. We propose that Centrobin might regulate microtubule nucleation and organization by controlling the amount of pericentriolar matrix.     

Human Ninein is a Centrosomal Autoantigen Recognized by CREST Patient Sera and Plays a Regulatory Role in Microtubule Nucleation

Centrosome is the major microtubule organizing center in mammalian cells that plays a critical role in a variety of cellular events by the microtubule arrays emanating from it. Despite its significance, the molecular mechanisms underlying the structure and function of the centrosome are still not clear. Herein we describe the identification of three isotypes of human ninein by expression library screening with autoimmune sera from CREST patients. All three ninein isotypes exhibit centrosomal localization throughout the cell cycle when GFP-tagged fusion proteins are expressed transiently in mammalian cells. Construction of serial deletions of GFP-tagged ninein reveals that a stretch of three leucine zippers with a flanking sequence is required and sufficient for centrosomal targeting. Overexpression of ninein results in mislocalization of ?-tubulin, recruiting it to ectopic (non-centrosomal) ninein-containing sites which are not active in nucleating microtubules. In these cells, nucleation of microtubules from the centrosome is also inhibited. These results thus suggest a regulatory role for ninein in microtubule nucleation.     

NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein gamma-tubulin. In mammals, gamma-tubulin associates with additional proteins into a large complex, the gamma-tubulin ring complex (gammaTuRC). We characterize NEDD1, a centrosomal protein that associates with gammaTuRCs. We show that the majority of gammaTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of gamma-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to gammaTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of gamma-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of gamma-tubulin to the centrosome.     

FBXL13 directs the proteolysis of CEP192 to regulate centrosome homeostasis and cell migration

Aberrant centrosome organisation with ensuing alterations of microtubule nucleation capacity enables tumour cells to proliferate and invade despite increased genomic instability. CEP192 is a key factor in the initiation process of centrosome duplication and in the control of centrosome microtubule nucleation. However, regulatory means of CEP192 have remained unknown. Here, we report that FBXL13, a binding determinant of SCF (SKP1‐CUL1‐F‐box)‐family E3 ubiquitin ligases, is enriched at centrosomes and interacts with the centrosomal proteins Centrin‐2, Centrin‐3, CEP152 and CEP192. Among these, CEP192 is specifically targeted for proteasomal degradation by FBXL13. Accordingly, induced FBXL13 expression downregulates centrosomal γ‐tubulin and disrupts centrosomal microtubule arrays. In addition, depletion of FBXL13 induces high levels of CEP192 and γ‐tubulin at the centrosomes with the consequence of defects in cell motility. Together, we characterise FBXL13 as a novel regulator of microtubule nucleation activity and highlight a role in promoting cell motility with potential tumour‐promoting implications.     

Cep70 promotes microtubule assembly in vitro by increasing microtubule elongation

Microtubules are dynamic cytoskeletal polymers present in all eukaryotic cells. In animal cells, they are organized by the centrosome, the major microtubule-organizing center. Many centrosomal proteins act coordinately to modulate microtubule assembly and organization. Our previous work has shown that Cep70, a novel centrosomal protein regulates microtubule assembly and organization in mammalian cells. However, the molecular details remain to be investigated. In this study, we investigated the molecular mechanism of how Cep70 regulates microtubule assembly using purified proteins. Our data showed that Cep70 increased the microtubule length without affecting the microtubule number in the purified system. These results demonstrate that Cep70 could directly regulate microtubule assembly by promoting microtubule elongation instead of microtubule nucleation.     

Characterization and Functional Aspects of Human Ninein Isoforms that Regulated by Centrosomal Targeting Signals and Evidence for Docking Sites to Direct Gamma-Tubulin

The functions of centrosomal protein ninein may be involved in microtubule minus end capping, centriole positioning, protein anchoring, and microtubule nucleation, but the true physiological function of various human hNinein isoforms remains to be determined. Here we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. These hNinein isoforms exhibit centrosomal (concentrated) and noncentrosomal (aggregated) localization when GFP-tagged fusion proteins are expressed transiently in mammalian cells. In a kinase assay, we show that the CCII region of hNinein provides a differential phosphorylation site by GSK3β. In addition, our data indicate that either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits γ-tubulin to centrosomal or non-centrosomal hNinein-containing sites, implying that the γ-tubulin localization may be hNinein-dependent. Further, our RNA interference experiment against all hNinein isoforms caused a significant decrease in the γ-tubulin signal in the centrosome. In domain swapping, we clearly show that the CCIIX-CCIIY region provides docking sites for γ-tubulin. Moreover, our data also show that nucleation of microtubules from the centrosome is significantly affected by the presence of either the full-length hNinein or CCIIX-CCIIY region overexpression. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit γ-tubulin at centrosomal and non-centrosomal sites.     

Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.     

Microtubule release from the centrosome in migrating cells

In migrating cells, force production relies essentially on a polarized actomyosin system, whereas the spatial regulation of actomyosin contraction and substrate contact turnover involves a complex cooperation between the microtubule (MT) and the actin filament networks (Goode, B.L., D.G. Drubin, and G. Barnes. 2000. Curr. Opin. Cell Biol., 12:63-71). Targeting and capture of MT plus ends at the cell periphery has been described, but whether or not the minus ends of these MTs are anchored at the centrosome is not known. Here, we show that release of short MTs from the centrosome is frequent in migrating cells and that their transport toward the cell periphery is blocked when dynein activity is impaired. We further show that MT release, but not MT nucleation or polymerization dynamics, is abolished by overexpression of the centrosomal MT-anchoring protein ninein. In addition, a dramatic inhibition of cell migration was observed; but, contrary to cells treated by drugs inhibiting MT dynamics, polarized membrane ruffling activity was not affected in ninein overexpressing cells. We thus propose that the balance between MT minus-end capture and release from the centrosome is critical for efficient cell migration.     

Polo-like kinase 1 regulates Nlp,a centrosome protein involved in microtubule nucleation

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.     

Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity

The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome.     

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.     

Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network

Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front.     

Localized RanGTP accumulation promotes microtubule nucleation at kinetochores in somatic mammalian cells

Centrosomes are the major sites for microtubule nucleation in mammalian cells, although both chromatin- and kinetochore-mediated microtubule nucleation have been observed during spindle assembly. As yet, it is still unclear whether these pathways are coregulated, and the molecular requirements for microtubule nucleation at kinetochore are not fully understood. This work demonstrates that kinetochores are initial sites for microtubule nucleation during spindle reassembly after nocodazole. This process requires local RanGTP accumulation concomitant with delocalization from kinetochores of the hydrolysis factor RanGAP1. Kinetochore-driven microtubule nucleation is also activated after cold-induced microtubule disassembly when centrosome nucleation is impaired, e.g., after Polo-like kinase 1 depletion, indicating that dominant centrosome activity normally masks the kinetochore-driven pathway. In cells with unperturbed centrosome nucleation, defective RanGAP1 recruitment at kinetochores after treatment with the Crm1 inhibitor leptomycin B activates kinetochore microtubule nucleation after cold. Finally, nascent microtubules associate with the RanGTP-regulated microtubule-stabilizing protein HURP in both cold- and nocodazole-treated cells. These data support a model for spindle assembly in which RanGTP-dependent abundance of nucleation/stabilization factors at centrosomes and kinetochores orchestrates the contribution of the two spindle assembly pathways in mammalian cells. The complex of RanGTP, the export receptor Crm1, and nuclear export signal-bearing proteins regulates microtubule nucleation at kinetochores.     

Reorganization of microtubule nucleation during muscle differentiation

Skeletal muscle differentiation involves a complete reorganization of the microtubule network. Nearly 20 years ago, Tassin et al. [1985: J Cell Biol 100:35-46] suggested a mechanism for this reorganization by showing a redistribution of the microtubule organizing center from the centrosome to the nuclear membrane. Little progress has been made since. It is still not clear whether centrosomal proteins are redistributed together, whether microtubules are nucleated at the nuclear membrane or transported there post-nucleation, and whether gamma-tubulin (gammatub) remains necessary for nucleation in myotubes. To investigate these questions, we have examined the redistribution of the centrosomal proteins pericentrin (PC), gammatub, and ninein in the C2 muscle cell line. Immunofluorescence of differentiated myotubes shows PC along the nuclear membrane whereas gammatub is only detected there after pre-fixation detergent extraction. After expression of a GFP-tagged gammatub, we observe a weak fluorescence along the nuclear membrane, confirming the presence of gammatub at a low concentration relative to PC. Microinjection of anti-gammatub antibodies into myotubes blocks microtubule growth from both nuclear membranes and centrosomal sites. The centrosomal microtubule-anchoring protein, ninein, is found at the nuclear membrane as well and its distribution appears independent of microtubule integrity. We conclude that centrosomal proteins are redistributed independently during muscle differentiation, to sites that nucleate microtubules both along the nuclear membranes and through the cytoplasm.     

Centrosome reduction during gametogenesis and its significance

Animal spermatids and primary oocytes initially have typical centrosomes comprising pairs of centrioles and pericentriolar fibrous centrosomal proteins. These somatic cell-like centrosomes are partially or completely degenerated during gametogenesis. Centrosome reduction during spermiogenesis comprises attenuation of microtubule nucleation function, loss of pericentriolar material, and centriole degeneration. Centrosome reduction during oogenesis is due to complete degeneration of centrioles, which leads to dispersal of the pericentriolar centrosomal proteins, loss of replicating capacity of the spindle poles, and switching to acentrosomal mode of spindle organization. Oocyte centrosome reduction plays an important role in preventing parthenogenetic embryogenesis and balancing centrosome number in the embryonic cells.     

The polarity protein Pard3 is required for centrosome positioning during neurulation

Microtubules are essential regulators of cell polarity, architecture and motility. The organization of the microtubule network is context-specific. In non-polarized cells, microtubules are anchored to the centrosome and form radial arrays. In most epithelial cells, microtubules are noncentrosomal, align along the apico-basal axis and the centrosome templates a cilium. It follows that cells undergoing mesenchyme-to-epithelium transitions must reorganize their microtubule network extensively, yet little is understood about how this process is orchestrated. In particular, the pathways regulating the apical positioning of the centrosome are unknown, a central question given the role of cilia in fluid propulsion, sensation and signaling. In zebrafish, neural progenitors undergo progressive epithelialization during neurulation, and thus provide a convenient in vivo cellular context in which to address this question. We demonstrate here that the microtubule cytoskeleton gradually transitions from a radial to linear organization during neurulation and that microtubules function in conjunction with the polarity protein Pard3 to mediate centrosome positioning. Pard3 depletion results in hydrocephalus, a defect often associated with abnormal cerebrospinal fluid flow that has been linked to cilia defects. These findings thus bring to focus cellular events occurring during neurulation and reveal novel molecular mechanisms implicated in centrosome positioning.     

CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the gamma-tubulin ring complex

Microtubule nucleation and organization by the centrosome require gamma-tubulin, a protein that exists in a macromolecular complex called the gamma-tubulin ring complex (gammaTuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including gamma-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the gammaTuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for gammaTuRC attachment to the centrosome but not for gammaTuRC assembly. Perturbing CDK5RAP2 function delocalized gamma-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in gammaTuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly.     

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.     

Centrosomal and non-centrosomal microtubules.

While microtubule (MT) arrays in cells are often focused at the centrosome, a variety of cell types contain a substantial number of non-centrosomal MTs. Epithelial cells, neurons, and muscle cells all contain arrays of non-centrosomal MTs that are critical for these cells' specialized functions. There are several routes by which non-centrosomal MTs can arise, including release from the centrosome, cytoplasmic assembly, breakage or severing, and stabilization from non-centrosomal sites. Once formed, MTs that are not tethered to the centrosome must be organized, which can be accomplished by means of self-organization or by capture and nucleation of MTs where they are needed. The presence of free MTs requires stabilization of minus ends, either by MT-associated proteins or by an end-capping complex. Although some of the basic elements of free MT formation and organization are beginning to be understood, a great deal of work is still necessary before we have a complete picture of how non-centrosomal MT arrays are assembled in specific cell types.