C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion

Abstract

The requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/ KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants. In contrast, oocytes with similarly severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 both negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and influences the dynamics of the central spindle-associated proteins Aurora B/AIR-2 and MgcRacGAP/CYK-4. We suggest that proper regulation by CLS-2 of both oocyte cortical stiffness and central spindle protein dynamics may influence contractile ring assembly during polar body extrusion in C. elegans oocytes.

C. elegans XMAP215/ZYG-9 and TACC/TAC-1 act at multiple times throughout oocyte meiotic spindle assembly to promote both the coalescence of pole foci into a bipolar structure, and the stability of coalesced poles, during oocyte meiotic cell division

“…Furthermore, the lack of a clear correlation between failed rotation and failed extrusion after metaphase upshifts in TS mutant oocytes, with polar body extrusion sometimes failing after apparently normal rotation, suggest that ZYG-9 and TAC-1 could have additional and more direct roles in polar body extrusion. Recent studies of the C. elegans TOG domain containing protein and CLASP2 family member CLS-2 indicate that proper regulation of microtubule stability is important for contractile ring assembly and ingression during polar body extrusion (Schlientz and Bowerman, 2020). However, in contrast to zyg-9 and tac-1 mutant oocytes, in which strong loss of function results in abnormally high levels of spindle- and cortex-associated microtubules during oocyte meiotic cell division, loss of CLS-2 results in reduced microtubule levels.…”

BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes

“…Cytoplasmic securin IFY-1 degradation proceeded at similar rates in wild-type and LET-92-depleted oocytes ( Figure 1H,I ; Figure 1—video 3 ), indicating that APC/C activity is largely unperturbed in the absence of PP2A. Given the similarity of this phenotype to the depletion of CLASP orthologue CLS-2 ( Dumont et al, 2010 ; Laband et al, 2017 ; Schlientz and Bowerman, 2020 ), we analysed the localisation of CLS-2 and detected no changes in CLS-2 levels throughout meiosis ( Figure 1—figure supplement 2 ; Figure 1—video 4 ).…”

“…During anaphase, chromosomes collapse into a small area where, in some cases, two chromosome masses were visible ( Figure 1C , yellow arrow). Interestingly, severe chromosome segregation defects do not necessarily lead to polar body extrusion (PBE) defects ( Schlientz and Bowerman, 2020 ). In the absence of PP2Ac, only 15% of oocytes achieved PBE ( Figure 1E , p<0.0001, Fisher’s exact test).…”

Cohesin is required for meiotic spindle assembly independent of its role in cohesion in C. elegans

“…We hypothesized that cohesin- dependent Aurora B on chromosomes might activate or inhibit microtubule-binding proteins that are required for coalescence of microtubule bundles or microtubule polymerization. Meiotic chromosome-associated spindle assembly factors include the katanin homolog, MEI-1 (Srayko, Buster, Bazirgan, McNally, & Mains, 2000), the kinesin-13, KLP-7 (Connolly, Sugioka, Chuang, Lowry, & Bowerman, 2015; Gigant et al, 2017), and the CLASP2 homolog, CLS-2 (Dumont et al, 2010; Schlientz & Bowerman, 2020). Loss of MEI-1 function results in apolar spindles with dispersed ASPM-1 (K. P. McNally & McNally, 2011) and reduced microtubule density (K. McNally, Audhya, Oegema, & McNally, 2006; Srayko, O’toole, Hyman, & Müller-Reichert, 2006) similar to those observed in cohesin mutants.…”

“…Loss of haspin-dependent CPC in this study caused a change in the localization pattern of KLP- 7 and CLS-2 on chromosomes. Loss of KLP-7 (Connolly et al, 2015; Gigant et al, 2017) or CLS-2 (Schlientz & Bowerman, 2020) results in multi-polar rather than apolar spindles but the effects of their over-expression or incorrect localization are not known. In Xenopus extracts, the primary function of Aurora B in spindle assembly has been suggested to be inhibition of the KLP-7 homolog, MCAK (Sampath et al, 2004).…”