The self-organized assembly of acentrosomal meiotic spindles has been extensively studied 1 but little is known about how chromosomes segregate on these spindles. Here, we investigate two chromosome-microtubule interaction mechanisms-kinetochores and chromokinesins-during meiosis in fertilized C. elegans oocytes. We show that the conserved kinetochore protein KNL-1 directs assembly of meiotic kinetochores that orient chromosomes on the acentrosomal spindles. However, in contrast to mitosis, chromosome separation during meiotic anaphase was kinetochore-independent. The chromokinesin KLP-19 did not contribute to chromosome orientation or anaphase, but stabilized late anaphase spindles. Prior to anaphase separation, meiotic kinetochores and spindle poles disassembled along with microtubules on the poleward side of the chromosomes; during anaphase, microtubules were formed between the separating chromosomes. Functional analysis implicated a set of proteins that localize to a ring-shaped domain between the kinetochores in pre-anaphase spindle assembly and anaphase separation. Ring domain proteins are localized by the chromosomal passenger complex (CPC), whose local enrichment is patterned by recombination to control step-wise loss of meiotic cohesion [2][3][4] . Thus, meiotic segregation in C. elegans is a two-stage process where kinetochores orient chromosomes but are dispensable for their separation. We suggest that separation is instead controlled by a meiosis-specific chromosomal domain to coordinate step-wise dissolution of cohesion with chromosome segregation. KeywordsMeiosis; Chromosome segregation; Kinetochore; Centromere; Bub1; Clasp; Microtubule; Spindle To study chromosome segregation on acentrosomal meiotic spindles, we used fertilized C. elegans oocytes because both female meiotic divisions and the first embryonic division can be monitored ex-utero. Assembly on a chromatin base containing the histone H3 variant CENP-A (CENtromeric Protein-A) is a universal feature of mitotic kinetochores that is conserved in C. elegans, despite the fact that it has holocentric chromosomes with kinetochores that run along along the length of each chromatid 5 . In contrast to CENP-Adirected mitotic assembly, a CENP-A-independent mechanism recruits kinetochore @ Corresponding author abdesai@ucsd.edu Phone: (858)-534-9698 Fax: (858)-534-7750 Address: CMM-E Rm 3052, 9500 Gilman Dr, La Jolla, CA 92093-0653. AUTHOR CONTRIBUTIONS All experimental data were generated by J.D., who also had primary responsibility for experimental design and data analysis. A.D. and K.O. contributed to experimental design and data analysis. J.D., A.D. and K.O. wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. In C. elegans, fertilization is followed by two rounds of meiotic chromosome segregation. During both meiotic divisions, the 6 chromosomes adopt a compact oval shape and kinetochore components accumulate on their surface in two opposing cup-like structures (Fig. 1a) separated by a ga...

Spindle formation is essential for stable inheritance of genetic material. Experiments in various systems indicate that Ran GTPase is crucial for meiotic and mitotic spindle assembly. Such an important role for Ran in chromatin-induced spindle assembly was initially demonstrated in Xenopus laevis egg extracts. However, the requirement of RanGTP in living meiotic cells has not been shown. In this study, we used a fluorescence resonance energy transfer probe to measure RanGTP-regulated release of importin β. A RanGTP-regulated gradient was established during meiosis I and was centered on chromosomes throughout mouse meiotic maturation. Manipulating levels of RanGTP in mice and X. laevis oocytes did not inhibit assembly of functional meiosis I spindles. However, meiosis II spindle assembly did not tolerate changes in the level of RanGTP in both species. These findings suggest that a mechanism common to vertebrates promotes meiosis I spindle formation in the absence of chromatin-induced microtubule production and centriole-based microtubule organizing centers.

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.

The ability to reproduce relies in most eukaryotes on specialized cells called gametes. Gametes are formed by the process of meiosis in which, after a single round of replication, two successive cell divisions reduce the ploidy of the genome. Fusion of gametes at fertilization reconstitutes diploidy. In most animal species, chromosome segregation during female meiosis occurs on spindles assembled in the absence of the major microtubule-organizing center, the centrosome. In mammals, oocyte meiosis is error-prone and underlies the majority of birth aneuploidies. Here, we review recent work on acentrosomal spindle formation and chromosome alignment/separation during oocyte meiosis in different animal models.

Summary To take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ~17s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the C. elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, though its GAP activity is only required until just prior to closure. Finally, the Chromosomal Passenger Complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation.

Summary Successive cell divisions during embryonic cleavage create increasingly smaller cells, so intracellular structures must adapt accordingly. Mitotic spindle size correlates with cell size, but the mechanisms for this scaling remain unclear. Using live cell imaging, we analyzed spindle scaling during embryo cleavage in the nematode Caenorhabditis elegans and sea urchin Paracentrotus lividus. We reveal a common scaling mechanism, where the growth rate of spindle microtubules scales with cell volume, which explains spindle shortening. Spindle assembly timing is however constant throughout successive divisions. Analyses in silico suggest that controlling the microtubule growth rate is sufficient to scale spindle length and maintain a constant assembly timing. We tested our in silico predictions to demonstrate that modulating cell volume or microtubule growth rate in vivo induces a proportional spindle size change. Our results suggest that scalability of the microtubule growth rate when cell size varies adapts spindle length to cell volume.

In animal cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Whereas mitotic kinases have been implicated in NEBD, how they coordinate their activity to trigger this event is unclear. Here, we show that both in human cells and Caenorhabditis elegans, the Polo-like kinase 1 (PLK-1) is recruited to the nuclear pore complexes, just prior to NEBD, through its Polo-box domain (PBD). We provide evidence that PLK-1 localization to the nuclear envelope (NE) is required for efficient NEBD. We identify the central channel nucleoporins NPP-1/Nup58, NPP-4/Nup54, and NPP-11/Nup62 as the critical factors anchoring PLK-1 to the NE in C. elegans. In particular, NPP-1, NPP-4, and NPP-11 primed at multiple Polo-docking sites by Cdk1 and PLK-1 itself physically interact with the PLK-1 PBD. We conclude that nucleoporins play an unanticipated regulatory role in NEBD, by recruiting PLK-1 to the NE thereby facilitating phosphorylation of critical downstream targets.

During cell division, spindle microtubules ensure an equal repartition of chromosomes between the two daughter cells. While the kinetochore-dependent mechanisms that drive mitotic chromosome segregation are well understood, in oocytes of most species atypical spindles assembled in absence of centrosomes entail poorly understood mechanisms of chromosome segregation. In particular, the structure(s) responsible for force generation during meiotic chromosome separation in oocytes is unclear. Using quantitative light microscopy, electron tomography, laser-mediated ablation, and genetic perturbations in the Caenorhabditis elegans oocyte, we studied the mechanism of chromosome segregation in meiosis. We find spindle poles are largely dispensable, and in fact act as brakes for chromosome segregation. Instead, our results suggest that CLS-2-dependent microtubules of the meiotic central spindle, located between the segregating chromosomes and aligned along the axis of segregation, are essential. Our results support a model in which inter-chromosomal microtubules of the central spindle push chromosomes apart during meiotic anaphase in oocytes.