Kinesin-1 prevents capture of the oocyte meiotic spindle by the sperm aster.
ABSTRACT: Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a nonmotor mechanism.
Project description:Fertilization occurs during female meiosis in most animals, which raises the question of what prevents the sperm DNA from interacting with the meiotic spindle. In this study, we find that Caenorhabditis elegans sperm DNA stays in a fixed position at the opposite end of the embryo from the meiotic spindle while yolk granules are transported throughout the embryo by kinesin-1. In the absence of F-actin, the sperm DNA, centrioles, and organelles were transported as a unit with the yolk granules, resulting in sperm DNA within 2 µm of the meiotic spindle. F-actin imaging revealed a cytoplasmic meshwork that might restrict transport in a size-dependent manner. However, increasing yolk granule size did not slow their velocity, and the F-actin moved with the yolk granules. Instead, sperm contents connect to the cortical F-actin to prevent interaction with the meiotic spindle.
Project description:Centrosome function in cell division requires their duplication, once, and only once, per cell cycle. Underlying centrosome duplication are alternating cycles of centriole assembly and separation. Work in vertebrates has implicated the cysteine protease separase in anaphase-coupled centriole separation (or disengagement) and identified this as a key step in licensing another round of assembly. Current models have separase cleaving a physical link between centrioles, potentially cohesin, that prevents reinitiation of centriole assembly unless disengaged. Here, we examine separase function in the C. elegans early embryo. We find that depletion impairs separation and consequently duplication of sperm-derived centrioles at the meiosis-mitosis transition. However, subsequent cycles proceed normally. Whereas mitotic centrioles separate in the context of cortical forces acting on a disassembling pericentriolar material, sperm centrioles are not associated with significant pericentriolar material or subject to strong forces. Increasing centrosomal microtubule nucleation restores sperm centriole separation and duplication in separase-depleted embryos, while forced pericentriolar material disassembly drives premature separation in mitosis. These results emphasize the critical role of cytoskeletal forces and the pericentriolar material in centriole separation. Separase contributes to separation where forces are limited, offering a potential explanation for results obtained in different experimental models.
Project description:The transition from meiosis to mitosis, classically defined by fertilization, is a fundamental process in development. However, its mechanism remains largely unexplored. In this paper, we report a surprising gradual transition from meiosis to mitosis over the first eight divisions of the mouse embryo. The first cleavages still largely share the mechanism of spindle formation with meiosis, during which the spindle is self-assembled from randomly distributed microtubule-organizing centers (MTOCs) without centrioles, because of the concerted activity of dynein and kinesin-5. During preimplantation development, the number of cellular MTOCs progressively decreased, the spindle pole gradually became more focused, and spindle length progressively scaled down with cell size. The typical mitotic spindle with centrin-, odf2-, kinesin-12-, and CP110-positive centrosomes was established only in the blastocyst. Overall, the transition from meiosis to mitosis progresses gradually throughout the preimplantation stage in the mouse embryo, thus providing a unique system to study the mechanism of centrosome biogenesis in vivo.
Project description: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.
Project description:Centriole duplication is of crucial importance during both mitotic and male meiotic divisions, but it is currently not known whether this process is regulated differently during the two modes of division. In Caenorhabditis elegans, the kinase ZYG-1 plays an essential role in both mitotic and meiotic centriole duplication. We have found that the C-terminus of ZYG-1 is necessary and sufficient for targeting to centrosomes and is important for differentiating mitotic and meiotic centriole duplication. Small truncations of the C-terminus dramatically lower the level of ZYG-1 at mitotic centrosomes but have little effect on the level of ZYG-1 at meiotic centrosomes. Interestingly, truncation of ZYG-1 blocks centrosome duplication in the mitotic cycle but leads to centrosome amplification in the meiotic cycle. Meiotic centriole amplification appears to result from the overduplication of centrioles during meiosis I and leads to the formation of multipolar meiosis II spindles. The extra centrioles also disrupt spermatogenesis by inducing the formation of supernumerary fertilization-competent spermatids that contain abnormal numbers of chromosomes and centrioles. Our data reveal differences in the regulation of mitotic and meiotic centrosome duplication, particularly with regard to ZYG-1 activity, and reveal an important role for centrosomes in spermatid formation.
Project description:Oocytes in humans, mice and other mammals lack identifiable centrioles. The proximal centriole brought in by the fertilizing sperm in humans and most other mammals appears to gives rise to the centrioles at the spindle poles in the zygote, and is believed to indicate that centrioles are inherited through the paternal lineage. However, both the proximal and distal sperm centrioles degenerate in mice and other rodents. A bipolar mitotic spindle nucleates from multiple centrosome-like structures in the mouse zygote and centrioles are not seen until the blastocyst stage, suggesting that centrioles are inherited through the maternal lineage in mice. We previously identified speriolin as a spermatogenic cell-specific binding partner of Cdc20 that co-localizes with pericentrin in mouse spermatocytes and is present in the centrosome in round spermatids.The nature and localization of speriolin in mouse and human sperm and the fate of speriolin following fertilization in the mouse were determined using immunofluorescence microscopy, immunoelectron microscopy and western blotting.Speriolin surrounds the intact proximal centriole in human sperm, but is localized at the periphery of the disordered distal centriole in mouse sperm. Human speriolin contains an internal 163-amino acid region not present in mouse that may contribute to localization differences. Speriolin is carried into the mouse oocyte during fertilization and remains associated with the decondensing sperm head in zygotes. The speriolin spot appears to undergo duplication or splitting during the first interphase and is detectable in 2-cell embryos.Speriolin is a novel centrosomal protein present in the connecting piece region of mouse and human sperm that is transmitted to the mouse zygote and can be detected throughout the first mitotic division.
Project description:Centrioles are evolutionarily conserved microtubule-based structures at the core of the animal centrosome that are essential for nucleating the axoneme of cilia. We hypothesized that centriole proteins have been under-represented in proteomic studies of the centrosome, because of the larger amount of pericentriolar material making up the centrosome. In this study, we have overcome this problem by determining the centriolar proteome of mammalian sperm cells, which have a pair of centrioles but little pericentriolar material. Mass spectrometry of sperm centrioles identifies known components of centrioles and many previously uncharacterized candidate centriole proteins. Assessment of localization of a subset of these candidates in cultured cells identified CCDC113, CCDC96, C4orf47, CCDC38, C7orf31, CCDC146, CCDC81 and CCDC116 as centrosome-associated proteins. We examined the highly conserved protein CCDC113 further and found that it is a component of centriolar satellites, is in a complex with the satellite proteins HAP1 and PCM1, and functions in primary cilium formation.
Project description:In many species where oocytes lack centrosomes, sperm contribute both genetic material and centriole(s) to the zygote. Correct centriole organization during male meiosis is critical to guarantee a normal bipolar mitotic spindle in the zygote. During Caenorhabditis elegans male meiosis, centrioles normally undergo two rounds of duplication, resulting in haploid sperm each containing a single tightly engaged centriole pair. Here we identify an unanticipated role for C. elegans HORMA (Hop1/Rev7/Mad2) domain proteins HTP-1/2 and HIM-3 in regulating centriole disengagement during spermatocyte meiosis. In him-3 and htp-1 htp-2 mutants, centrioles separate inappropriately during meiosis II, resulting in spermatids with disengaged centrioles. Moreover, extra centrosomes are detected in a subset of zygotes. Together, these data implicate HIM-3 and HTP-1/2 in preventing centriole disengagement during meiosis II. We showed previously that HTP-1/2 prevents premature loss of sister chromatid cohesion during the meiotic divisions by inhibiting removal of meiotic cohesin complexes containing the REC-8 subunit. Worms lacking REC-8, or expressing a mutant separase protein with elevated local concentration at centrosomes and in sperm, likewise exhibit inappropriate centriole separation during spermatocyte meiosis. These observations are consistent with HIM-3 and HTP-1/2 preventing centriole disengagement by inhibiting separase-dependent cohesin removal. Our data suggest that the same specialized meiotic mechanisms that function to prevent premature release of sister chromatid cohesion during meiosis I in C. elegans also function to inhibit centriole separation at meiosis II, thereby ensuring that the zygote inherits the appropriate complement of chromosomes and centrioles.
Project description:Centrosomes are composed of two centrioles surrounded by pericentriolar material (PCM). However, the sperm and the oocyte modify or lose their centrosomes. Consequently, how the zygote establishes its first centrosome, and in particular, the origin of the second zygotic centriole, is uncertain. Drosophila melanogaster spermatids contain a single centriole called the Giant Centriole (GC) and a Proximal centriole-like (PCL) structure whose function is unknown. We found that, like the centriole, the PCL loses its protein markers at the end of spermiogenesis. After fertilization, the first two centrioles are observed via the recruitment of the zygotic PCM proteins and are seen in asterless mutant embryos that cannot form centrioles. The zygote's centriolar proteins label only the daughter centrioles of the first two centrioles. These observations demonstrate that the PCL is the origin for the second centriole in the Drosophila zygote and that a paternal centriole precursor, without centriolar proteins, is transmitted to the egg during fertilization.
Project description:Centrosomes are dynamic organelles that consist of a pair of cylindrical centrioles, surrounded by pericentriolar material. The pericentriolar material contains factors that are involved in microtubule nucleation and organization, and its recruitment varies during the cell cycle. We report here that proteasome inhibition in HeLa cells induces the accumulation of several proteins at the pericentriolar material, including gamma-tubulin, GCP4, NEDD1, ninein, pericentrin, dynactin, and PCM-1. The effect of proteasome inhibition on centrosome proteins does not require intact microtubules and is reversed after removal of proteasome inhibitors. This accrual of centrosome proteins is paralleled by accumulation of ubiquitin in the same area and increased polyubiquitylation of nonsoluble gamma-tubulin. Cells that have accumulated centrosome proteins in response to proteasome inhibition are impaired in microtubule aster formation. Our data point toward a role of the proteasome in the turnover of centrosome proteins, to maintain proper centrosome function.