A kinetochore-independent mechanism drives anaphase chromosome separation during acentrosomal meiosis

Abstract

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...

The Chromosomal Passenger Complex Is Required for Meiotic Acentrosomal Spindle Assembly and Chromosome Biorientation

“…Some previous studies have suggested that chromosome alignment depends on lateral interactions during acentrosomal meiosis (Brunet et al 1999;Schuh and Ellenberg 2007;Wignall and Villeneuve 2009;Walczak et al 2010). However, an alternative model incorporates an important role for kinetochore microtubules (Dumont et al 2010). Kinetochore microtubules in oocytes have been inferred by Hughes et al (2011) and could be the cold-resistant karyosome-associated microtubules that we have observed (Salmon and Begg 1980;Rieder 1981).…”

“…(Rogers et al 2002;Wignall and Villeneuve 2009). The C. elegans CPC ring is a complex structure that, as in Drosophila, contains motor proteins (Klp-19) (Powers et al 2004) and is required for segregation of homologs at meiosis I (Wignall and Villeneuve 2009;Dumont et al 2010). The importance of noncentromeric CPC in a variety of organisms suggests that the unique demands of acentrosomal meiosis have resulted in a meiosis-specific CPC/central spindle localization pattern with a conserved role in spindle assembly and chromosome segregation.…”

Polar body emission

“…On the other hand, the holocentric C. elegans oocytes appear to employ lateral microtubules to align chromosomes in metaphase [Wignall and Villeneuve, 2009] and segregate chromosomes independently of kinetochores [Dumont et al, 2010].…”

The eggshell in the C. elegans oocyte‐to‐embryo transition

“…Kinetochore attachment and centrosomedependent pulling forces are required to segregate sister chromatids to opposite poles during mitosis (Cheeseman and Desai, 2008;Lampson and Cheeseman, 2011;Tanaka, 2010). Centrosomes are absent in the meiotic spindle and separation during anaphase is driven by pushing forces arising between the sister chromatids (Dumont et al, 2010). However, kinetochores are still important to orient chromosomes on the meiotic spindle, as depletion of kinetochore proteins such as KNL-1 results in a lagging chromosome phenotype (Dumont et al, 2010).…”

“…Centrosomes are absent in the meiotic spindle and separation during anaphase is driven by pushing forces arising between the sister chromatids (Dumont et al, 2010). However, kinetochores are still important to orient chromosomes on the meiotic spindle, as depletion of kinetochore proteins such as KNL-1 results in a lagging chromosome phenotype (Dumont et al, 2010). Search-and-capture of kinetochores by remodeling microtubules is the primary means by which chromosomes are linked to the spindle.…”