Microtubules are required for centrosome expansion and positioning while microfilaments are required for centrosome separation in sea urchin eggs during fertilization and mitosis

Centrosomes undergo cell cycle-dependent changes in shape and separations, changes that govern the organization of the cytoskeleton. The cytoskeleton is largely organized by the centrosome; however, this investigation explores the importance of cytoskeletal elements in directing centrosome shape. Since the sea urchin egg during fertilization and mitosis displays dramatic and synchronous changes in centrosome shape, the effects of cytoskeletal inhibitors on centrosome compaction, expansion, and separation were explored by the use of anticentrosome immunofluorescence microscopy. Centrosome expansion and separation was studied during two phases: the transition after sperm incorporation, when the compact sperm centrosome enlarges and the sperm aster develops, and from prometaphase to telophase, when the compact spindle poles enlarge. Compaction was investigated when the dispersed centrosome at interphase condenses into the two spindle poles at prometaphase. Although centrosome expansion and separation typically occur concurrently, beta-mercaptoethanol results in centrosome separation independent of expansion. Microtubule inhibitors prevent centrosome expansion and separation, and expanded centrosomes collapse. Since pronuclear union is arrested by microtubule inhibitors, this treatment also affords the opportunity to explore the relative attractiveness of the male and female pronuclei for these centrosomal antigens. Both pronuclei acquire centrosomal material; though only the male centrosome is capable of organizing a functional bipolar mitotic apparatus at first division, the female centrosome nucleates a monaster. Microfilament inhibition (cytochalasin D) prevents centrosome separation but not expansion or compaction. These results demonstrate that as the centrosome shapes the cytoskeleton, the cytoskeleton alters centrosome shape.     

Centrosome-intrinsic mechanisms modulate centrosome integrity during fever

The centrosome is critical for cell division, ciliogenesis, membrane trafficking, and immunological synapse function. The immunological synapse is part of the immune response, which is often accompanied by fever/heat stress (HS). Here we provide evidence that HS causes deconstruction of all centrosome substructures primarily through degradation by centrosome-associated proteasomes. This renders the centrosome nonfunctional. Heat-activated degradation is centrosome selective, as other nonmembranous organelles (midbody, kinetochore) and membrane-bounded organelles (mitochondria) remain largely intact. Heat-induced centrosome inactivation was rescued by targeting Hsp70 to the centrosome. In contrast, Hsp70 excluded from the centrosome via targeting to membranes failed to rescue, as did chaperone inactivation. This indicates that there is a balance between degradation and chaperone rescue at the centrosome after HS. This novel mechanism of centrosome regulation during fever contributes to immunological synapse formation. Heat-induced centrosome inactivation is a physiologically relevant event, as centrosomes in leukocytes of febrile patients are disrupted.     

Microtubule nucleation and release from the neuronal centrosome

We have proposed that microtubules (MTs) destined for axons and dendrites are nucleated at the centrosome within the cell body of the neuron, and are then released for translocation into these neurites (Baas, P. W., and H. C. Joshi. 1992. J. Cell Biol. 119:171-178). In the present study, we have tested the capacity of the neuronal centrosome to act as a generator of MTs for relocation into other regions of the neuron. In cultured sympathetic neurons undergoing active axonal outgrowth, MTs are present throughout the cell body including the region around the centrosome, but very few (< 10) are directly attached to the centrosome. These results indicate either that the neuronal centrosome is relatively inactive with regard to MT nucleation, or that most of the MTs nucleated at the centrosome are rapidly released. Treatment for 6 h with 10 micrograms/ml nocodazole results in the depolymerization of greater than 97% of the MT polymer in the cell body. Within 5 min after removal of the drug, hundreds of MTs have assembled in the region of the centrosome, and most of these MTs are clearly attached to the centrosome. A portion of the MTs are not attached to the centrosome, but are aligned side-by-side with the attached MTs, suggesting that the unattached MTs were released from the centrosome after nucleation. In addition, unattached MTs are present in the cell body at decreasing levels with increasing distance from the centrosome. By 30 min, the MT array of the cell body is indistinguishable from that of controls. The number of MTs attached to the centrosome is once again diminished to fewer than 10, suggesting that the hundreds of MTs nucleated from the centrosome after 5 min were subsequently released and translocated away from the centrosome. These results indicate that the neuronal centrosome is a highly potent MT- nucleating structure, and provide strong indirect evidence that MTs nucleated from the centrosome are released for translocation into other regions of the neuron.     

A stereoscopic analysis of the centrosome structure in the cells of continuous and primary cell cultures]

A 3D reconstruction of the centrosome region was made based on series of semithick sections in tissue culture cells. It was shown that: 1) the total number of microtubules attached to the centrosome is about 30-50 of which only 20% or less run farther than 2 microns away from the centrosome; 2) a certain number of short microtubules (less than 1 micron length) is present in the vicinity of the centrosome, the majority of them are attached to the centrosome; 3) many microtubules around the centrosome have no direct contact with either centrioles, or other microtubule-convergent structures; 4) the majority of free microtubules are comparatively long (more than 1 micron length); 5) almost all the microtubules running closer than 2 microns to the centrosome are oriented towards it with their proximal ends. The radial distribution of free microtubules around the centrosome support the supposition that they may appear as a result of their detachment from the microtubule-nucleating centres.     

Centrosome positioning in polarized cells: Common themes and variations

The centrosome position is tightly regulated during the cell cycle and during differentiated cellular functions. Because centrosome organizes the microtubule network to coordinate both intracellular organization and cell signaling, centrosome positioning is crucial to determine either the axis of cell division, the direction of cell migration or the polarized immune response of lymphocytes. Since alteration of centrosome positioning seems to promote cell transformation and tumor spreading, the molecular mechanisms controlling centrosome movement in response to extracellular and intracellular cues are under intense investigation. Evolutionary conserved pathways involving polarity proteins and cytoskeletal rearrangements are emerging as common regulators of centrosome positioning in a wide variety of cellular contexts.     

Causes and consequences of centrosome abnormalities in cancer

Centrosome amplification is a hallmark of cancer. However, despite significant progress in recent years, we are still far from understanding how centrosome amplification affects tumorigenesis. Boveri''s hypothesis formulated more than 100 years ago was that aneuploidy induced by centrosome amplification promoted tumorigenesis. Although the hypothesis remains appealing 100 years later, it is also clear that the role of centrosome amplification in cancer is more complex than initially thought. Here, we review how centrosome abnormalities are generated in cancer and the mechanisms cells employ to adapt to centrosome amplification, in particular centrosome clustering. We discuss the different mechanisms by which centrosome amplification could contribute to tumour progression and the new advances in the development of therapies that target cells with extra centrosomes.     

Centrosome inheritance in starfish zygotes: selective loss of the maternal centrosome after fertilization

The mature egg inherits a centrosome from the second meiotic spindle, and the sperm introduces a second centrosome at fertilization. Since only one of these centrosomes survives to be used in development, specific mechanisms must exist to control centrosome inheritance. To investigate how centrosome inheritance is controlled we used starfish eggs as a model system, because they undergo meiosis after fertilization. As a result, the fate of the maternal and paternal centrosomes can be followed by light microscopy and experimentally manipulated in vivo. We show initially that only the paternal centrosome is used in starfish zygote development; the maternal centrosome retained from meiosis II is functionally lost before first mitosis. We then tested a number of possible ways in which the zygote could exert this differential control over the stability of centrosomes initially residing in the same cytoplasm. The results of these experiments can be summarized as follows: (1) Although the microtubule organizing center activity of the maternal centrosome is not degraded after meiosis, the ability of this centrosome to double at successive mitoses is lost. (2) The sperm centrosome is not "masked" from cytoplasmic conditions which could destabilize all centrosomes during or after the meiotic sequence. (3) The functional loss of the maternal centrosome is not due to its cortical location. (4) The loss of this doubling capacity is determined by the egg, not by putative inhibitory factors from the fertilizing sperm. (5) The destabilization of the maternal centrosome is not due to the complete loss of its centrioles. Together, these results demonstrate that all maternal centrosomes are equivalent and that they are intrinsically different from the paternal centrosome. This intrinsic difference, in concert with a change in cytoplasmic conditions after meiosis, determines the selective loss of the maternal centrosome inherited from the meiosis II spindle.     

CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity

Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin‐dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. Results. In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N‐ and C‐terminal regions of CDC25B and requires CDC25B binding to its CDK—cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. Conclusions. Our results therefore suggest that CDC25B associates with a Ctn2‐containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components.     

Centrosome-associated NDR kinase regulates centrosome duplication

Human NDR kinases are upregulated in some cancer types, yet their functions still remain undefined. Here, we report the first known function of a mammalian NDR kinase by demonstrating that human NDR directly contributes to centrosome duplication. A subpopulation of endogenous NDR localizes to centrosomes in a cell-cycle-dependent manner. Overexpression of NDR resulted in centrosome overduplication in a kinase-activity-dependent manner, while expression of kinase-dead NDR or depletion of NDR by small interfering RNA (siRNA) negatively affected centrosome duplication. By targeting NDR to the centrosome, we show that the centrosomal pool of NDR is sufficient to generate supernumerary centrosomes. Furthermore, our data indicate that NDR-driven centrosome duplication requires Cdk2 activity and that Cdk2-induced centrosome amplification is affected upon reduction of NDR activity. Overall, considering that centrosome overduplication is linked to cellular transformation, our observations may also provide a molecular link between mammalian NDR kinases and cancer.     

Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.     

Cell cycle-dependent localization of monoclonal antibodies raised against isolated Dictyostelium centrosomes.

The ultrastructure of the Dictyostelium centrosome is markedly different from that of the well known yeast spindle pole body and vertebrate centriole-containing centrosome. It consists of a box-shaped, layered core structure surrounded by a corona with dense nodules embedded in an amorphous matrix. For further structural and biochemical analyses of this type of centrosome we used highly enriched isolated Dictyostelium centrosomes as an antigen to raise 14 new centrosomal monoclonal antibodies. Immunofluorescence microscopy and Western blot analysis revealed that at least 10 of them were directed against different antigens. Immunofluorescence microscopy also showed that the monoclonal antibodies fell into three different groups: A) antibodies localizing to the centrosome during the entire cell cycle; B) antibodies staining the centrosome mainly during mitosis; and C) antibodies labeling centrosome associated structures. All antibodies, except one, exhibited a cell cycle-dependent staining pattern underscoring the highly dynamic properties of the Dictyostelium centrosome.     

Centrosome docking at the immunological synapse is controlled by Lck signaling

Docking of the centrosome at the plasma membrane directs lytic granules to the immunological synapse. To identify signals controlling centrosome docking at the synapse, we have studied cytotoxic T lymphocytes (CTLs) in which expression of the T cell receptor-activated tyrosine kinase Lck is ablated. In the absence of Lck, the centrosome is able to translocate around the nucleus toward the immunological synapse but is unable to dock at the plasma membrane. Lytic granules fail to polarize and release their contents, and target cells are not killed. In CTLs deficient in both Lck and the related tyrosine kinase Fyn, centrosome translocation is impaired, and the centrosome remains on the distal side of the nucleus relative to the synapse. These results show that repositioning of the centrosome in CTLs involves at least two distinct steps, with Lck signaling required for the centrosome to dock at the plasma membrane.     

A tentative classification of centrosome abnormalities in cancer

Centrosome anomalies are detected in virtually all human cancers. They have been implicated in multipolar mitoses, chromosome missegregation, and genomic instability. Despite extensive studies on the type and frequency of centrosome anomalies, a causative relationship between centrosome aberrations and chromosomal instability has been difficult to establish. For example, centrosome amplification can be present without associated chromosomal instability. In addition, not all cells appear to be permissive for centrosome-related mitotic defects suggesting that cellular mechanisms that limit the harmful effects of spindle malformation on genome integrity may exist. This review proposes to classify centrosome abnormalities in tumor cells into three groups based on their relevance to genomic instability: primary centrosome overduplication, transient centrosome accumulation, and permanent centrosome accumulation. Whereas the first two categories are associated with an increased risk of chromosomal missegregation, the latter category may not contribute to the propagation of genomic instability. Therefore, centrosome anomalies should not per se be viewed as a universal cause of chromosomal instability, rather, they need to be assessed in the cellular context in which they occur.     

A subset of centrosomal proteins are arranged in a tubular conformation that is reproduced during centrosome duplication

The centrosome plays a fundamental role in organizing the interphase cytoskeleton and the mitotic spindle, and its protein complexity is modulated to support these functions. The centrosome must also duplicate itself once during each cell cycle, thus ensuring the formation of a bipolar spindle and its continuity through successive cell divisions. In this study, we have used a battery of antibodies directed against centrosomal components to study the general organization of the centrosome during the cell cycle and during the centrosome duplication process. We demonstrate that a subset of centrosomal proteins are arranged together to form a tubular pattern within the centrosome. The tubular conformation defined by these proteins has a polarity and is closed at one end. The centriole complement of the centrosome is normally placed near this end. We show that the "wall" of the tube is enriched in proteins such as CDC2, ninein, and pericentrin as well as gamma-tubulin. In addition, a subset of gamma-tubulin is localized to the "lumen" of the tube. We also demonstrate, for the first time, that antibody staining can be used to detect centrosome duplication allowing the identification of duplication intermediates. We show that one product of centrosome duplication is the replication of the tubular structure found within the centrosome. The position of the centriole duplexes prior to and during centrosome duplication is documented and a model of the morphogenesis of the centrosome during the duplication process is proposed.     

Fine structural studies of the bipolarization of the mitotic apparatus in the fertilized sea urchin egg. I. The structure and behavior of centrosomes before fusion of the pronuclei

During fertilization the sperm brings two centrosomes into the egg. One centrosome contains a centriole of normal length originally seen as the basal body of the sperm flagellum. Characteristically, the proximal half is enwrapped in osmiophilic material. This centrosome is attached to the centrosomal fossa, a bowl-shaped depression of the nuclear envelope of the male pronucleus. Microtubules radiate out from the osmiophilic half characterizing this structure as a centrosome and microtubule organizing center (MTOC). The second centrosome which also acts as an MTOC is attached to the mitochondrion of the sperm. At the beginning it appears as an unstructured accumulation of osmiophilic material out of which later on centriolar microtubules grow. Though this centrosome is marked by an immature centriole it is capable of organizing microtubules and of reproducing itself. This centrosome becomes loosely associated with the female pronucleus by means of microtubules. Then it separates from the mitochondrion which finally is lost. When the two pronuclei fuse, the centrosome derived from the basal body remains firmly attached to the centrosomal fossa, which has persisted in the envelope of the zygote nucleus after pronuclear fusion. Using the fossa as a marker of the position of this centrosome on the nuclear surface, we conclude that it is a stationary centrosome in the process of bipolarization for the first mitosis.     

Structural centrosome aberrations promote non‐cell‐autonomous invasiveness

Centrosomes are the main microtubule‐organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein‐like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP‐induced structural centrosome aberrations trigger the escape (“budding”) of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP‐induced centrosome aberrations trigger a re‐organization of the cytoskeleton, which stabilizes microtubules and weakens E‐cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non‐cell‐autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes.     

中心体在配子与早期胚胎中的遗传模式及其在辅助生殖中的意义

中心体由中心粒周围物质(PCM)围绕一对相互垂直的圆柱形中心粒组成,是哺乳动物细胞内主要的微管组织中心,在细胞分裂时发挥重要的作用。中心体以半保留的形式复制,在精子及卵母细胞发生时会发生减灭,精子和卵母细胞各保留部分中心体的成分,在受精后重新组成功能完整中心体。精子的中心体结构发生异常将会导致男性的不育,卵母细胞的老化也会引起中心体蛋白缺陷,从而产生纺锤体结构异常,并导致受精及早期胚胎发育异常。中心体的结构与功能,与人类受精及胚胎发育相关密切,在辅助生殖中具有重要意义。     

The mouse Mps1p-like kinase regulates centrosome duplication.

The yeast Mps1p protein kinase acts in centrosome duplication and the spindle assembly checkpoint. We demonstrate here that a mouse Mps1p ortholog (esk, which we designate mMps1p) regulates centrosome duplication. Endogenous mMps1p and overexpressed GFP-mMps1p localize to centrosomes and kinetochores in mouse cells. Overexpression of GFP-mMps1p causes reduplication of centrosomes during S phase arrest. In contrast, a kinase-deficient mutant blocks centrosome duplication altogether. Control of centrosome duplication by mMps1p requires a known regulator of the process, Cdk2. Inhibition of Cdk2 prevents centrosome reduplication and destabilizes mMps1p, causing its subsequent loss from centrosomes, suggesting that Cdk2 promotes mMps1p's centrosome duplication function by regulating its stability during S phase. Thus, mMps1p, an in vitro Cdk2 substrate, regulates centrosome duplication jointly with Cdk2.