Involvement of a centrosomal protein kendrin in the maintenance of centrosome cohesion by modulating Nek2A kinase activity

Centrosome cycle is strictly coordinated with chromosome duplication cycle to ensure the faithful segregation of chromosomes. Centrosome duplication occurs from the beginning of S phase, and the duplicated centrosomes are held together by centrosome cohesion to function as a single microtubule organizing center during interphase. At late G2 phase centrosome cohesion is disassembled by Nek2A kinase-mediated phosphorylation and, as a consequence, centrosomes are split and constitute spindle poles in mitosis. It has been reported that depletion of a centrosomal protein kendrin (also named pericentrin) induces premature centrosome splitting in interphase, however, it remains unknown how kendrin contributes to the maintenance of centrosome cohesion. Here we show that kendrin associates with Nek2A kinase, which exhibits considerably low activity. Nek2A kinase activity is inhibited in vitro by addition of the Nek2A-binding region of kendrin in a dose-dependent manner. Furthermore, ectopic expression of the same region decreases the number of the cells with split centrosomes at late G2 phase. Taken together, these results suggest that kendrin anchors Nek2A and suppresses its kinase activity at the centrosomes, and thus, is involved in the mechanism to prevent premature centrosome splitting during interphase.     

The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity

Formation of a bipolar spindle is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, defects in centrosome number and structural organization can lead to a loss of bipolarity. In addition, microtubule-mediated pulling and pushing forces acting on centrosomes and chromosomes are also important for bipolar spindle formation. Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase that has essential roles in the formation of a bipolar spindle with focused poles. However, the mechanism by which Plk1 regulates spindle-pole formation is poorly understood. Here, we identify a novel centrosomal substrate of Plk1, Kizuna (Kiz), depletion of which causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in multipolar spindles. We demonstrate that Kiz is critical for establishing a robust mitotic centrosome architecture that can endure the forces that converge on the centrosomes during spindle formation, and suggest that Plk1 maintains the integrity of the spindle poles by phosphorylating Kiz.     

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.     

Mitochondrial genome regulates mitotic fidelity by maintaining centrosomal homeostasis

Centrosomes direct spindle morphogenesis to assemble a bipolar mitotic apparatus to enable error-free chromosome segregation and preclude chromosomal instability (CIN). Amplified centrosomes, a hallmark of cancer cells, set the stage for CIN, which underlies malignant transformation and evolution of aggressive phenotypes. Several studies report CIN and a tumorigenic and/or aggressive transformation in mitochondrial DNA (mtDNA)-depleted cells. Although several nuclear-encoded proteins are implicated in centrosome duplication and spindle organization, the involvement of mtDNA encoded proteins in centrosome amplification (CA) remains elusive. Here we show that disruption of mitochondrial function by depletion of mtDNA induces robust CA and mitotic aberrations in osteosarcoma cells. We found that overexpression of Aurora A, Polo-like kinase 4 (PLK4), and Cyclin E was associated with emergence of amplified centrosomes. Supernumerary centrosomes in rho0 (mtDNA-depleted) cells resulted in multipolar mitoses bearing “real” centrosomes with paired centrioles at the multiple poles. This abnormal phenotype was recapitulated by inhibition of respiratory complex I in parental cells, suggesting a role for electron transport chain (ETC) in maintaining numeral centrosomal homeostasis. Furthermore, rho0 cells displayed a decreased proliferative capacity owing to a G₂/M arrest. Downregulation of nuclear-encoded p53 in rho0 cells underscores the importance of mitochondrial and nuclear genome crosstalk and may perhaps underlie the observed mitotic aberrations. By contrast, repletion of wild-type mtDNA in rho0 cells (cybrid) demonstrated a much lesser extent of CA and spindle multipolarity, suggesting partial restoration of centrosomal homeostasis. Our study provides compelling evidence to implicate the role of mitochondria in regulation of centrosome duplication, spindle architecture, and spindle pole integrity.     

Centrobin: a novel daughter centriole-associated protein that is required for centriole duplication

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The pair of centrioles, which are the core components of the centrosome, duplicate once per cell cycle. Centrosomes play a pivotal role in orchestrating the formation of the bipolar spindle during mitosis. Recent studies have linked centrosomal activity on centrioles or centriole-associated structures to cytokinesis and cell cycle progression through G1 into the S phase. In this study, we have identified centrobin as a centriole-associated protein that asymmetrically localizes to the daughter centriole. The silencing of centrobin expression by small interfering RNA inhibited centriole duplication and resulted in centrosomes with one or no centriole, demonstrating that centrobin is required for centriole duplication. Furthermore, inhibition of centriole duplication by centrobin depletion led to impaired cytokinesis.     

Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.     

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.     

The Drosophila wispy gene is required for RNA localization and other microtubule-based events of meiosis and early embryogenesis

RNAs are localized by microtubule-based pathways to both the anterior and posterior poles of the developing Drosophila oocyte. We describe a new gene, wispy, required for localization of mRNAs to both poles of the egg. Embryos from wispy mothers arrest development after abnormal oocyte meiosis and failure of pronuclei to fuse. Our analysis of spindle and chromosome movements during meiosis reveals defects in spindle structures correlated with very high frequencies of chromosome nondisjunction and loss. Spindle defects include abnormally shaped spindles, spindle spurs, and ectopic spindles associated with lost chromosomes, as well as mispositioning of the meiosis II spindles. The polar body nuclei do not associate with their normal monastral arrays of microtubules, the sperm aster is reduced in size, and the centrosomes often dissociate from a mitotic spindle that forms in association with the male pronucleus. We show that wispy is required to recruit or maintain known centrosomal proteins with two types of microtubule organizing centers (MTOCs): (1) the central MTOC that forms between the meiosis II tandem spindles and (2) the centrosomes of the mitotic spindle. We propose that the wispy gene product functions directly in several microtubule-based events in meiosis and early embryogenesis and speculate about its possible mode of action.     

Polo-like kinase 1 independently controls microtubule-nucleating capacity and size of the centrosome

Centrosomes are composed of a centriolar core surrounded by a pericentriolar material (PCM) matrix that docks microtubule-nucleating γ-tubulin complexes. During mitotic entry, the PCM matrix increases in size and nucleating capacity in a process called centrosome maturation. Polo-like kinase 1 (PLK1) is recruited to centrosomes and phosphorylates PCM matrix proteins to drive their self-assembly, which leads to PCM expansion. Here, we show that in addition to controlling PCM expansion, PLK1 independently controls the generation of binding sites for γ-tubulin complexes on the PCM matrix. Selectively preventing the generation of PLK1-dependent γ-tubulin docking sites led to spindle defects and impaired chromosome segregation without affecting PCM expansion, highlighting the importance of phospho-regulated centrosomal γ-tubulin docking sites in spindle assembly. Inhibiting both γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulated the effects of loss of centrosomal PLK1 on the ability of centrosomes to catalyze spindle assembly.     

Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.     

Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner

During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.     

Peripheral, non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells

In centrosome-containing cells, microtubules utilized in spindle formation are thought to be nucleated at the centrosome. However, spindle formation can proceed following experimental destruction of centrosomes or in cells lacking centrosomes, suggesting that non-centrosome-associated microtubules may contribute to spindle formation, at least when centrosomes are absent. Direct observation of prometaphase cells expressing GFP-alpha-tubulin shows that peripheral, non-centrosome-associated microtubules are utilized in spindle formation, even in the presence of centrosomes. Clusters of peripheral microtubules moved into the centrosomal region, demonstrating that a centrosomal microtubule array can be composed of both centrosomally nucleated and peripheral microtubules. Peripheral bundles also moved laterally into the forming spindle between the spindle poles; 3D reconstructions of fixed cells reveal interactions between peripheral and centrosome-associated microtubules. The spindle pole component NuMA and gamma-tubulin were present at the foci of peripheral microtubule clusters, indicating that microtubules moved into the spindle with minus ends leading. Photobleach- and photoactivation-marking experiments of cells expressing GFP-tubulin or a photoactivatable variant of GFP-tubulin, respectively, demonstrate that microtubule motion into the forming spindle results from transport and sliding interactions, not treadmilling. Our results directly demonstrate that non-centrosome-associated microtubules contribute to spindle formation in centrosome-containing cells.     

Mechanisms of spindle-pole organization are influenced by kinetochore activity in mammalian cells

The spindle is a fusiform bipolar-microtubule array that is responsible for chromosome segregation during mitosis. Focused poles are an essential feature of spindles in vertebrate somatic cells, and pole focusing has been shown to occur through a centrosome-independent self-organization mechanism where microtubule motors cross-link and focus microtubule minus ends. Most of our understanding of this mechanism for pole focusing derives from studies performed in cell-free extracts devoid of centrosomes and kinetochores. Here, we examine how sustained force from kinetochores influences the mechanism of pole focusing in cultured cells. We show that the motor-driven self-organization activities associated with NuMA (i.e., cytoplasmic dynein) and HSET are not necessary for pole focusing if sustained force from kinetochores is inhibited in Nuf2- or Mis12-deficient cells. Instead, pole organization relies on TPX2 as it cross-links spindle microtubules to centrosome-associated mitotic asters. Thus, both motor-driven and static-cross-linking mechanisms contribute to spindle-pole organization, and kinetochore activity influences the mechanism of spindle-pole organization. The motor-driven self-organization of microtubule minus ends at spindle poles is needed to organize spindle poles in vertebrate somatic cells when kinetochores actively exert force on spindle microtubules.     

Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.     

Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which “amplifies” spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle.     

The centrosome cycle in the mitotic cycle of sea urchin eggs

When sea urchin eggs entering mitosis are exposed to an appropriate concentration of mercaptoethanol, the chromosome cycle is restrained while the centrosome cycle advances. The two poles of the mitotic apparatus separate into four poles, while the chromosomes remain in their metaphase arrangements until released by the removal of the mercaptoethanol. We follow the centrosomes through the stages of the generation of two poles by each original pole. In electron microscopic studies, the osmiophilic component of the centrosomes serves as an indicator of their changing forms as each pole generates two poles. In light microscopic studies, including observations of birefringence, the shapes of the polar ends of the spindles are taken as indicators of the shapes of the centrosomes. The successive stages of the centrosome cycle are (1) compact spherical centrosomes at the time of formation of the mitotic apparatus; (2) expansion and flattening of the centrosomes, leading to (3) formation of thin flat plates, perpendicular to the spindle axis. Corresponding to the extended flat shape of the centrosomes, the spindle poles are flat; microtubules 'point' to the centrosomal plate and not the centrioles. The centrioles are separated in the flattening of the centrosomes. (4) The flat plate divides into two and each of the two halves becomes more compact, defining two separate poles. Our findings resurrect and update Boveri's [5] observations and interpretations of the centrosome. Centrosomes have shapes. The shapes may be imparted to the microtubular structures that they generate. The formation of two separate centrosomes from one, in the formation of mitotic poles, is describable as a sequence of changes in shape.     

Spindle pole centrosomes of sea urchin embryos are partially composed of material recruited from maternal stores

The spindle poles of fertilized sea urchin eggs have commonly been modeled as being derived from the centrosomes of the fertilizing spermatozoon. Boveri's theory of fertilization, proposed at the turn of the century, states that the maternal centrosome is suppressed or inactivated during oogenesis and that the sperm centrosome is functionally dominant. In support of this proposal, more recent studies have shown that the sperm imports a determinant that is involved in centrosomal replication. Examination of sea urchin zygotes immunofluorescently labeled with a new anti-centrosomal antibody by quantitative confocal laser-scanning microscopy shows, however, that spindle pole centrosomes are not exclusively paternal structures, but additionally contain material derived from maternal pools. Furthermore, this maternal centrosomal material is divided among daughter blastomeres during cleavage. It therefore appears that although the sperm centrosome plays a dominant role in organizing the spindle poles, much of the centrosomal material within the spindle poles of the zygote is actually recruited from preexisting egg cytoplasmic stores. These data indicate that centrosomes of sea urchin embryos are biparentally derived, composite organelles.     

csi2p modulates microtubule dynamics and organizes the bipolar spindle for chromosome segregation

Proper chromosome segregation is of paramount importance for proper genetic inheritance. Defects in chromosome segregation can lead to aneuploidy, which is a hallmark of cancer cells. Eukaryotic chromosome segregation is accomplished by the bipolar spindle. Additional mechanisms, such as the spindle assembly checkpoint and centromere positioning, further help to ensure complete segregation fidelity. Here we present the fission yeast csi2⁺. csi2p localizes to the spindle poles, where it regulates mitotic microtubule dynamics, bipolar spindle formation, and subsequent chromosome segregation. csi2 deletion (csi2Δ) results in abnormally long mitotic microtubules, high rate of transient monopolar spindles, and subsequent high rate of chromosome segregation defects. Because csi2Δ has multiple phenotypes, it enables estimates of the relative contribution of the different mechanisms to the overall chromosome segregation process. Centromere positioning, microtubule dynamics, and bipolar spindle formation can all contribute to chromosome segregation. However, the major determinant of chromosome segregation defects in fission yeast may be microtubule dynamic defects.     

The microtubule nucleation activity of centrobin in both the centrosome and cytoplasm

Centrobin resides in daughter centriole and play a critical role in centriole duplication. Nucleation and stabilization of microtubules are known biological activities of centrobin. Here, we report a specific localization of centrobin outside the centrosome. Centrobin was associated with the stable microtubules. In hippocampal cells, centrobin formed cytoplasmic dots in addition to the localization at both centrosomes with the mother and daughter centrioles. Such specific localization pattern suggests that cytoplasmic centrobin is not just a reserved pool for centrosomal localization but also has a specific role in the cytoplasm. In fact, centrobin enhanced microtubule formation outside as well as inside the centrosome. These results propose specific roles of the cytoplasmic centrobin for noncentrosomal microtubule formation in specific cell types and during the cell cycle.     

CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.