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.     

The role of centrosomes in fertilization,cell division and establishment of asymmetry during embryo development

Centrosomes play significant central roles during reproduction, cell division, embryo development and stem cell biology, and a wealth of new information has been accumulated during the past decade that spans molecular details and newly discovered functions/dysfunctions for specific centrosome proteins in various cellular activities. The present review will focus on the current state of knowledge on the role of germ cell centrosomes during fertilization, formation of the zygote centrosome, centrosome duplication and separation during first embryonic cell division, and asymmetric cell divisions during cell differentiation and subsequent embryo development. It will also address asymmetric cell division in stem cells and formation of the primary cilium during embryo development.     

Centriole inheritance

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.Key words: centrosome, centriol, spindle, mitosis, microtubule, cell cycle, checkpoints     

Behaviour of centrosomes in early Tubifex embryos: asymmetric segregation and mitotic cycle-dependent duplication

An antibody raised against a highly conserved peptide of -tubulin (Joshi et al. 1992) recognized a 50 kDa polypeptide in centrosomes in Tubifex embryos. Centrosomes labelled with this antibody are found at both poles of the first meiotic spindle and at the inner pole of the second meiotic spindle. At the transition to the second meiosis, there is no change in morphology of the centrosomes which are retained in the egg proper. In contrast, as the second meiosis proceeds from anaphase to telophase, centrosomes labelled with the antibody gradually become smaller, but are still recognized as tiny dots; each egg exhibits no more than one tiny dot. The first cleavage spindles exhibit a centrosome at one pole but not at the other. The spindle pole with a centrosome forms an aster which is inherited by the larger cell, CD, of the two-cell embryo; the centrosome-free spindle pole then becomes anastral and is segregated to a smaller cell AB. Centrosomes are present in the C and D cell lineages but not in the A and B lineages, at least up to the eighth cleavage cycle. During cleavage stages, centrosomes duplicate early in telophase of each mitosis, and their size changes in a cell cycle-specific fashion. Centrosomes which otherwise duplicate asynchronously in separate cells do so synchronously in a common cytoplasm. Centrosome duplication is inhibited by nocodazole but not by cytochalasin D. An examination of embryos treated with cycloheximide or aphidicolin also suggests that centrosome duplication during cleavages requires protein synthesis but no DNA replication per se. These results suggest that the centrosome cycle in Tubifex blastomeres is linked to the mitotic cycle more closely than is that in other animals.     

Free centrosomes: Where do they all come from?

Centrosomes act as major microtubule-organizing centers in most cell types. Their functions in interphase and mitosis are usually facilitated by their association with the nucleus. This may be particularly true in very large cells. Several papers report free centrosomes in syncytial Drosophila embryos. However, this phenotype often remains little explored. Yet, free centrosomes can occur by multiple mechanisms, including functional defects of the mitotic spindle, detachment of centrosomes from the nuclear envelope, centrosome inactivation upon DNA damage, and de novo centrosome genesis. Deciphering the cellular mechanism leading to free centrosomes upon a given perturbation such as a mutation or injection of a drug, can provide valuable clues regarding the nature of the molecular pathway affected. To this end, genetic and cytological tests, as well as time-lapse imaging are available. These studies can inform on the biology of centrosomes, cell cycle regulation and cytoskeletal dynamics. Here we briefly discuss what to make of free centrosomes in the fly embryo.     

HOPS is an essential constituent of centrosome assembly

Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, γ-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer.     

Evolutionary problems in centrosome and centriole biology

Centrosomes have been an enigma to evolutionary biologists. Either they have been the subject of ill‐founded speculation or they have been ignored. Here, we highlight evolutionary paradoxes and problems of centrosome and centriole evolution and seek to understand them in the light of recent advances in centrosome biology. Most evolutionary accounts of centrosome evolution have been based on the hypothesis that centrosomes are replicators, independent of the nucleus and cytoplasm. It is now clear, however, that this hypothesis is not tenable. Instead, centrosomes are formed de novo each cell division, with the presence of an old centrosome regulating, but not essential for, the assembly of a new one. Centrosomes are the microtubule‐organizing centres of cells. They can potentially affect sensory and motor characters (as the basal body of cilia), as well as the movements of chromosomes during cell division. This latter role does not seem essential, however, except in male meiosis, and the reasons for this remain unclear. Although the centrosome is absent in some taxa, when it is present, its structure is extraordinarily conserved: in most taxa across eukaryotes, it does not appear to evolve at all. And yet a few insect groups display spectacular hypertrophy of the centrioles. We discuss how this might relate to the unusual reproductive system found in these insects. Finally, we discuss why the fate of centrosomes in sperm and early embryos might differ between different groups of animals.     

Occludin localizes to centrosomes and modifies mitotic entry

Proper control of cell cycle progression and barrier function are essential processes to the maintenance of epithelial cell homeostasis. The contribution of tight junction proteins to barrier function is well established, whereas their contribution to cell cycle control is only beginning to be understood. Centrosomes are the principal microtubule organizing centers in eukaryotic cells and centrosome duplication and separation are linked to the cell cycle and mitotic entry. Here we demonstrate that occludin localizes with centrosomes in Madin-Darby canine kidney cells. Immunocytochemistry and biochemical fractionation studies reveal occludin localizes with centrosomes during interphase and occludin Ser-490 phosphorylation at centrosomes increases with mitotic entry. Stable expression of aspartic acid phosphomimetic (S490D) results in centrosomal localization of occludin and increases cell numbers. Furthermore, we provide evidence that occludin regulates centrosome separation and mitotic entry as the nonphosphorylatable alanine mutation (S490A) impedes centrosome separation, delays mitotic entry, and reduces proliferation. Collectively, these studies demonstrate a novel location and function for occludin in centrosome separation and 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.     

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

Remodeling of donor cell centrosomes and the centrosome-associated cytoskeleton is crucially important for nuclear cloning as centrosomes are the main microtubule organizing centers that play a significant role in cell division and embryo development. Centrosome dysfunctions have been implicated in various diseases including cancer and metabolic disorders and may also play a role in developmental abnormalities that are frequently seen in cloned animals. In the present studies we investigated microtubule organization and the reorganization and fate of the integral centrosome protein γ-tubulin and the centrosome-associated protein centrin in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using antibodies to γ-tubulin or GFP-centrin transfected mouse fibroblasts as donor cells. Microtubules were stained with antibodies to α-tubulin. In-vitro-fertilized oocytes and nuclear transfer (NT) reconstructed oocytes were sequentially analyzed at different developmental stages. Epi-fluorescence results revealed mitotic spindle abnormalities in NT embryos during the first cell cycle (39.4%, 13/33) which were significantly higher than those in IVF embryos (17.0%, 7/41). The abnormalities in IVF embryos are due to polyspermy while the abnormalities in NT embryos are due to donor cell centrosome dysfunctions. In the NT embryos with abnormal microtubule and centrosome organization, γ-tubulin staining revealed multipolar centrosome foci while DAPI staining showed misalignment of chromosomes. In intraspecies and interspecies embryos the GFP-centrin signal was detected until 3 hrs after fusion. GFP-centrin was not detected at 8 hrs after NT which is consistent with previous results using anti-centrin antibody staining in intraspecies NT porcine embryos. These data indicate that 1) abnormalities in microtubule and centrosome organization are associated with nuclear cloning at a higher rate than observed in IVF embryos; 2) centrosome and cytoskeletal abnormalities in IVF embryos are due to polyspermy while centrosome and cytoskeletal abnormalities in NT embryos are due to donor cell centrosome dysfunctions; and 3) GFP-centrin of the donor cell centrosome provides a reliable marker to follow its fate in intraspecies reconstructed embryos.     

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.     

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.     

Centrosome function in normal and tumor cells

Centrosomes nucleate microtubules that form the mitotic spindle and regulate the equal division of chromosomes during cell division. In cancer, centrosomes are often found amplified to greater than two per cell, and these tumor cells frequently have aneuploid genomes. In this review, we will discuss the cellular factors that regulate the proper duplication of the centrosome and how these regulatory steps can lead to abnormal centrosome numbers and abnormal mitoses. In particular, we highlight the newly emerging role of the Breast Cancer 1 (BRCA1) ubiquitin ligase in this process.     

Hook2 localizes to the centrosome, binds directly to centriolin/CEP110 and contributes to centrosomal function

Centrosomes serve as microtubule-organizing centers. However, centrosome function depends on microtubule organization and protein transport because the formation, positioning and maintenance of centrosomes require microtubule-dependent retrograde transport. Linker proteins that associate with the motor protein dynein, organelles and microtubules facilitate loading of cargos for retrograde transport and thus contribute to the composition and placement of the centrosome and other juxtanuclear protein complexes. Members of the hook family of proteins may function as adaptors to link various organelle cargos to dynein for transport and have also been implicated directly in centrosome positioning. Here, we show that mammalian hook2, a previously uncharacterized member of the hook family, localizes to the centrosome through all phases of the cell cycle, the C-terminal domain of hook2 directly binds to centriolin/CEP110, the expression of the C-terminal domain of centriolin/CEP110 alters the distribution of endogenous hook2 and mislocalized wild-type or mutant hook2 proteins perturb endogenous centrosomal and pericentrosomal proteins in cultured mammalian cells. In addition, interference with hook2 function results in the loss of the radial organization of microtubules and a defect in regrowth of microtubules following their nocodazole-induced depolymerization. Thus, we propose that hook2 contributes to the establishment and maintenance of centrosomal structure and function.     

Centrosomes: The good and the bad for brain development

Centrosomes nucleate and organise the microtubule cytoskeleton in animal cells. These membraneless organelles are key structures for tissue organisation, polarity and growth. Centrosome dysfunction, defined as deviation in centrosome numbers and/or structural integrity, has major impact on brain size and functionality, as compared with other tissues of the organism. In this review, we discuss the contribution of centrosomes to brain growth during development. We discuss in particular the impact of centrosome dysfunction in Drosophila and mammalian neural stem cell division and fitness, which ultimately underlie brain growth defects.     

Modification of Cul1 regulates its association with proteasomal subunits

Accurate transmission of chromosomes from parent to progeny cell requires assembly of a bipolar spindle. Centrosomes (spindle pole body in yeast) are critical for the biogenesis of this complex mitotic apparatus since they confer bipolarity on the spindle and serve as the site of microtubule polymerization. In each division cycle, the centrosome is duplicated and the sister-centrosomes move away from each other, forming the two poles of the spindle. While the structure and the duplication of centrosomes have been investigated extensively, the understanding of the control of their segregation remains scant. Recent findings are beginning to yield insights into the regulation of centrosome segregation in yeast and its link to the mitotic kinase.     

A coiled‐coil protein is required for coordination of karyokinesis and cytokinesis in Toxoplasma gondii

Toxoplasma gondii is a unicellular eukaryotic pathogen that belongs to the Apicomplexa phylum, which encompasses some of the deadliest pathogens of medical and veterinary importance. The centrosome is key to the organisation and coordination of the cell cycle and division of apicomplexan parasites. The T. gondii centrosome possesses a particular bipartite structure (outer and inner cores). One of the main roles of the centrosome is to ensure proper coordination of karyokinesis. However, how these 2 events are coordinated is still unknown in T. gondii, for which the centrosome components are poorly described. To gain more insights into the biology and the composition of the T. gondii centrosome, we characterised a protein that resides at the interface of the outer and inner core centrosomes. TgCep530 is a large coiled‐coil protein with an essential role in the survival of the parasite. Depletion of this protein leads to the accumulation of parasites lacking nuclei and disruption of the normal cell cycle. Lack of TgCep530 results in a discoordination between the nuclear cycle and the budding cycle that yields fully formed parasites without nuclei. TgCep530 has a crucial role in the coordination of karyokinesis and cytokinesis.     

The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1–cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Δ-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase–anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary “licensing” event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.     

A non-canonical mode of microtubule organization operates throughout pre-implantation development in mouse

In dividing animal cells, the centrosome, comprising centrioles and surrounding pericentriolar-material (PCM), is the major interphase microtubule-organizing center (MTOC), arranging a polarized array of microtubules (MTs) that controls cellular architecture. The mouse embryo is a unique setting for investigating the role of centrosomes in MT organization, since the early embryo is acentrosomal, and centrosomes emerge de novo during early cleavages. Here we use embryos from a GFP::CETN2 transgenic mouse to observe the emergence of centrosomes and centrioles in embryos, and show that unfocused acentriolar centrosomes first form in morulae (~16–32-cell stage) and become focused at the blastocyst stage (~64–128 cells) concomitant with the emergence of centrioles. We then used high-resolution microscopy and dynamic tracking of MT growth events in live embryos to examine the impact of centrosome emergence upon interphase MT dynamics. We report that pre-implantation mouse embryos of all stages employ a non-canonical mode of MT organization that generates a complex array of randomly oriented MTs that are preferentially nucleated adjacent to nuclear and plasmalemmal membranes and cell-cell interfaces. Surprisingly, however, cells of the early embryo continue to employ this mode of interphase MT organization even after the emergence of centrosomes. Centrosomes are found at MT-sparse sites and have no detectable impact upon interphase MT dynamics. To our knowledge, the early embryo is unique among proliferating cells in adopting an acentrosomal mode of MT organization despite the presence of centrosomes, revealing that the transition to a canonical mode of interphase MT organization remains incomplete prior to implantation.     

Separate to operate: control of centrosome positioning and separation

The centrosome is the main microtubule (MT)-organizing centre of animal cells. It consists of two centrioles and a multi-layered proteinaceous structure that surrounds the centrioles, the so-called pericentriolar material. Centrosomes promote de novo assembly of MTs and thus play important roles in Golgi organization, cell polarity, cell motility and the organization of the mitotic spindle. To execute these functions, centrosomes have to adopt particular cellular positions. Actin and MT networks and the association of the centrosomes to the nuclear envelope define the correct positioning of the centrosomes. Another important feature of centrosomes is the centrosomal linker that connects the two centrosomes. The centrosome linker assembles in late mitosis/G1 simultaneously with centriole disengagement and is dissolved before or at the beginning of mitosis. Linker dissolution is important for mitotic spindle formation, and its cell cycle timing has profound influences on the execution of mitosis and proficiency of chromosome segregation. In this review, we will focus on the mechanisms of centrosome positioning and separation, and describe their functions and mechanisms in the light of recent findings.