Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.     

CEP90 Is Required for the Assembly and Centrosomal Accumulation of Centriolar Satellites,Which Is Essential for Primary Cilia Formation

Centriolar satellites are PCM-1-positive granules surrounding centrosomes. Proposed functions of the centriolar satellites include protein targeting to the centrosome, as well as communication between the centrosome and surrounding cytoplasm. CEP90 is a centriolar satellite protein that is critical for spindle pole integrity in mitotic cells. In this study, we examined the biological functions of CEP90 in interphase cells. CEP90 physically interacts with PCM-1 at centriolar satellites, and this interaction is essential for centrosomal accumulation of the centriolar satellites and eventually for primary cilia formation. CEP90 is also required for BBS4 loading on centriolar satellites and its localization in primary cilia. Our results imply that the assembly and transport of centriolar satellites are critical steps for primary cilia formation and ciliary protein recruitment.     

Spindle pole fragmentation due to proteasome inhibition

During interphase, the centrosome concentrates cell stress response molecules, including chaperones and proteasomes, into a proteolytic center. However, whether the centrosome functions as proteolytic center during mitosis is not known. In this study, cultured mammalian cells were treated with the proteasome inhibitor MG 132 and spindle morphology in mitotic cells was characterized in order to address this issue. Proteasome inhibition during mitosis leads to the formation of additional asters that cause the assembly of multipolar spindles. The cause of this phenomenon was investigated by inhibiting microtubule-based transport and protein synthesis. These experimental conditions prevented the formation of supernumerary asters during mitosis. In addition, the expression of dsRed without proteasome inhibition led to the fragmentation of spindle poles. These experiments showed that the formation of extra asters depends on intact microtubule-based transport and protein synthesis. These results suggest that formation of supernumerary asters is due to excessive accumulation of proteins at the spindle poles and consequently fragmentation of the centrosome. Together, this leads to the conclusion that the centrosome functions as proteolytic center during mitosis and proteolytic activity at the spindle poles is necessary for maintaining spindle pole integrity.     

Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment

Mitotic spindles assemble from two centrosomes, which are major microtubule‐organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an “inside‐out” mechanism, ending with establishment of the poles. We used HSET (kinesin‐14) as a tool to shift meiotic spindle assembly toward a mitotic “outside‐in” mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic‐like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique “inside‐out” mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes.     

The effect of the UV microirradiation of the centrosome on cell behavior. III. The ultrastructure of the centrosome after irradiation]

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam in metaphase or in anaphase. Electron microscopy showed that immediately after irradiation the microtubules around the centrosome were maintained, and that the ultrastructure of both irradiated and nonirradiated poles was similar. After microirradiation of the centrosome in metaphase, the mitotic halo around this centrosome was retained, but in due time the number of microtubules was getting less compared to that around the nonirradiated centrosome. When daughter cells with irradiated centrosomes are passing into the interphase, their centrioles are not separated from each other, no primary cilia are formed, and no replication of centrioles occurs. In the interphase cells with irradiated centrosomes, satellites are formed on the active centriole, but centrosome-attached microtubules are practically absent.     

Functional role of centrosomes in spindle assembly and organization

The centrosome is the main MT organizing center in animal cells, and has traditionally been regarded as essential for organization of the bipolar spindle that facilitates chromosome segregation during mitosis. Centrosomes are associated with the poles of the mitotic spindle, and several cell types require these organelles for spindle formation. However, most plant cells and some female meiotic systems get along without this organelle, and centrosome‐independent spindle assembly has now been identified within some centrosome containing cells. How can such observations, which point to mutually incompatible conclusions regarding the requirement of centrosomes in spindle formation, be interpreted? With emphasis on the functional role of centrosomes, this article summarizes the current models of spindle formation, and outlines how observations obtained from spindle assembly assays in vitro may reconcile conflicting opinions about the mechanism of spindle assembly. It is further described how Drosophila mutants are used to address the functional interrelationships between individual centrosomal proteins and spindle formation in vivo. © 2004 Wiley‐Liss, Inc.     

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform.     

Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, gamma-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. gamma-tubulin translocated into the two poles of the transient spindles, while no accumulated gamma-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, gamma-tubulin was translocated to the spindle poles. The distribution of gamma-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. gamma-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as pericentriolar material surrounding a pair of centrioles, is degraded in the 1-cell reconstituted embryos after activation; (2) components of donor cell centrosomes contribute to the formation of the transient spindle and normal functional mitotic spindle, although the contribution of centrosomal material stored in the recipient ooplasm is not excluded; and (3) components of donor cell centrosomes involved in spindle assembly may not be species-specific.     

Requirements for NuMA in maintenance and establishment of mammalian spindle poles

Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.     

The centrosome and bipolar spindle assembly

In vertebrate somatic cells the centrosome functions as the major microtubule-organizing center (MTOC), which splits and separates to form the poles of the mitotic spindle. However, the role of the centriole-containing centrosome in the formation of bipolar mitotic spindles continues to be controversial. Cells normally containing centrosomes are still able to build bipolar spindles after their centrioles have been removed or ablated. In naturally occurring cellular systems that lack centrioles - such as plant cells and many oocytes - bipolar spindles form in the complete absence of canonical centrosomes. These observations have led to the notion that centrosomes play no role during mitosis. However, recent work has re-examined spindle assembly in the absence of centrosomes, both in cells that naturally lack them, and those that have had them experimentally removed. The results of these studies suggest that an appreciation of microtubule network organization- both before and after nuclear envelope breakdown (NEB) - is the key to understanding the mechanisms that regulate spindle assembly and the generation of bipolarity.     

The centrosome and bipolar spindle assembly: Does one have anything to do with the other?

In vertebrate somatic cells, the centrosome functions as the major microtubule-organizing center (MTOC), which splits and separates to form the poles of the mitotic spindle. However, the role of the centriole-containing centrosome in the formation of bipolar mitotic spindles continues to be controversial. Cells normally containing centrosomes are still able to build bipolar spindles after their centrioles have been removed or ablated. In naturally occurring cellular systems that lack centrioles, such as plant cells and many oocytes, bipolar spindles form in the complete absence of canonical centrosomes. These observations have led to the notion that centrosomes play no role during mitosis. However, recent work has re-examined spindle assembly in the absence of centrosomes, both in cells that naturally lack them and those that have had them experimentally removed. The results of these studies suggest that an appreciation of microtubule network organization, both before and after nuclear envelope breakdown (NEB), is the key to understanding the mechanisms that regulate spindle assembly and the generation of bipolarity.Key words: centrosome, centriole, mitosis, spindle, cell cycle, meiosis, plant cell, microsurgery     

Centrin depletion causes cyst formation and other ciliopathy-related phenotypes in zebrafish

Most bona fide centrosome proteins including centrins, small calcium-binding proteins, participate in spindle function during mitosis and play a role in cilia assembly in non-cycling cells. Although the basic cellular functions of centrins have been studied in lower eukaryotes and vertebrate cells in culture, phenotypes associated with centrin depletion in vertebrates in vivo has not been directly addressed. To test this, we depleted centrin2 in zebrafish and found that it leads to ciliopathy phenotypes including enlarged pronephric tubules and pronephric cysts. Consistent with the ciliopathy phenotypes, cilia defects were observed in differentiated epithelial cells of ciliated organs such as the olfactory bulb and pronephric duct. The organ phenotypes were also accompanied by cell cycle deregulation namely mitotic delay resulting from mitotic defects. Overall, this work demonstrates that centrin2 depletion causes cilia-related disorders in zebrafish. Moreover, given the presence of both cilia and mitotic defects in the affected organs, it suggests that cilia disorders may arise from a combination of these defects.     

Centrin depletion causes cyst formation and other ciliopathy-related phenotypes in zebrafish

Most bona fide centrosome proteins, including centrins, small calcium-binding proteins, participate in spindle function during mitosis and play a role in cilia assembly in non-cycling cells. Although the basic cellular functions of centrins have been studied in lower eukaryotes and vertebrate cells in culture, phenotypes associated with centrin depletion in vertebrates in vivo has not been directly addressed. To test this, we depleted centrin2 in zebrafish and found that it leads to ciliopathy phenotypes, including enlarged pronephric tubules and pronephric cysts. Consistent with the ciliopathy phenotypes, cilia defects were observed in differentiated epithelial cells of ciliated organs, such as the olfactory bulb and pronephric duct. The organ phenotypes were also accompanied by cell cycle deregulation, namely, mitotic delay resulting from mitotic defects. Overall, this work demonstrates that centrin2 depletion causes cilia-related disorders in zebrafish. Moreover, given the presence of both cilia and mitotic defects in the affected organs, it suggests that cilia disorders may arise from a combination of these defects.Key words: centrosome, cilia, centrin, mitosis, cystogenesis, ciliopathies, zebrafish     

TBCCD1, a new centrosomal protein,is required for centrosome and Golgi apparatus positioning

In animal cells the centrosome is positioned at the cell centre in close association with the nucleus. The mechanisms responsible for this are not completely understood. Here, we report the first characterization of human TBCC‐domain containing 1 (TBCCD1), a protein related to tubulin cofactor C. TBCCD1 localizes at the centrosome and at the spindle midzone, midbody and basal bodies of primary and motile cilia. Knockdown of TBCCD1 in RPE‐1 cells caused the dissociation of the centrosome from the nucleus and disorganization of the Golgi apparatus. TBCCD1‐depleted cells are larger, less efficient in primary cilia assembly and their migration is slower in wound‐healing assays. However, the major microtubule‐nucleating activity of the centrosome is not affected by TBCCD1 silencing. We propose that TBCCD1 is a key regulator of centrosome positioning and consequently of internal cell organization.     

Cep192 and the generation of the mitotic spindle

The cellular mechanisms used to generate sufficient microtubule polymer mass to drive the assembly and function of the mitotic spindle remain a matter of great interest. As the primary microtubule nucleating structures in somatic animal cells, centrosomes have been assumed to figure prominently in spindle assembly. At the onset of mitosis, centrosomes undergo a dramatic increase in size and microtubule nucleating capacity, termed maturation, which is likely a key event in mitotic spindle formation. Interestingly, however, spindles can still form in the absence of centrosomes calling into question the specific mitotic role of these organelles. Recent work has shown that the human centrosomal protein, Cep192, is required for both centrosome maturation and spindle assembly thus providing a molecular link between these two processes. In this article, we propose that Cep192 does so by forming a scaffolding on which proteins involved in microtubule nucleation are sequestered and become active in mitotic cells. Normally, this activity is largely confined to centrosomes but in their absence continues to function but is dispersed to other sites within the cell.     

LEGOs® and legacies of centrioles and centrosomes

Centriole construction, now revealed by crystallography, proteomics, and imaging to be a sophisticated assembly of interlocking bricks, resembles LEGOs—albeit centrioles have remarkable dynamic capabilities, including self‐assembly and dis‐assembly, kinases and post‐translational modifications, self‐replication, and still mysterious mechanisms for transmission through each cell cycle and via the gametes during development. Centrioles are created by core proteins that aggregate to form unique ninefold‐symmetrical paracrystalline cylinders. The centrosome then coalesces as a cloud of pericentriolar material (PCM) around the centriole. Together they comprise the cell's microtubule organizing center (MTOC), which governs the shape, functions, and dynamics of the cell's microtubule (MT) arrays. This includes the meiotic and mitotic spindle apparatus for chromosome segregation, the accuracy of which is crucial for avoiding aneuploidies and resulting cancer, birth defects, or infertility. Centrioles’ replication and transmission mechanisms—and reduplication blocks—across cell cycles and generations, are only now becoming tractable to molecular analysis, which allows research to address questions about spindle assembly with neither centrioles nor centrosomes or de novo centriole formation. Here we discuss the latest insights into centriole and centrosome assembly and function and their transgenerational inheritance.     

Donor centrosome regulation of initial spindle formation in mouse somatic cell nuclear transfer: roles of gamma-tubulin and nuclear mitotic apparatus protein 1

During the process of spindle-chromosome complex depletion in the oocyte, it is unclear whether both gamma-tubulin and nuclear mitotic apparatus protein 1 (NUMA1), which are required for mitotic organization and spindle assembly, are removed. The role of the donor cell centrosome and donor nuclear NUMA1 in the initial spindle morphogenesis and chromosome remodeling also remains unclear. In the present study, we show that in the mouse, the level of gamma-tubulin in the poles and around the metaphase II spindle declines significantly, whereas only approximately 10% of NUMA1 is removed during spindle-chromosome complex depletion in the recipient oocyte. This process does not impede initial spindle morphogenesis and is regulated by the centrosome of the donor cumulus cell. Retaining the donor cell centrosome establishes a monopolar spindle, whereas prior removal of the centrosome by a narrow-bore micropipette leads to bipolar spindle formation. Our data show that the centrosome of the donor cell regulates initial spindle morphogenesis and that the donor cumulus cell NUMA1 compensates for the deficiency in recipient NUMA1 during the formation of metaphase-like structures after nuclear transfer. Full-term offspring of cloned mice were obtained after injection of donor cells only with a pipette having an inner diameter of 7-8 microm, which retained the donor cell centrosome. In contrast, removing the donor cell centrosome with a small pipette impaired preimplantation development and prevented full-term development. In conclusion, the initial spindle assembly of a metaphase-like spindle is regulated by the centrosome from the donor cell in the mouse.     

Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.     

The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and gamma-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.     

Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest

Centrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).