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.