Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex

Abstract

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole.

Inhibition of ectopic microtubule assembly by the kinesin-13 KLP-7 prevents chromosome segregation and cytokinesis defects in oocytes

“…The defects that we observed in chromosome segregation and PBE in KLP-7-depleted or -deleted oocytes prompted us to analyze anaphase I central spindle organization and function, this spindle being essential for both chromosome segregation and PBE in the C. elegans oocyte Fabritius et al, 2011). Deconvolution microscopy on fixed oocytes in anaphase I showed obvious central spindle microtubule organization defects in KLP-7-depleted oocytes (Fig.…”

“…The precise mechanism of this atypical segregation is still unclear but involves microtubule-dependent forces exerted on chromosomes Muscat et al, 2015;McNally et al, 2016). The third specific adaptation of oocyte meiosis is polar body extrusion (PBE), which corresponds to an extremely asymmetric partitioning of the oocyte creating a tiny polar body with most of the cytoplasm maintained in the oocyte (Zhang et al, 2008;Dorn et al, 2010;Fabritius et al, 2011;Maddox et al, 2012).…”

Polar body cytokinesis

“…During mitosis, centralspindlin localizes to the region of microtubule plus end overlap at the center of the spindle midzone and, together with the microtubule bundling protein PRC1 SPD-1 , organizes the midzone. Centralspindlin also colocalizes with PRC1 SPD-1 on meiotic central spindles in C. elegans but in striking contrast to mitosis, all three components are dispensable for assembling the meiotic spindle midzone [Powers et al, 1998;Dumont et al, 2010;Fabritius et al, 2011]. However, centralspindlin is essential for PBE, suggesting that its main function in oocytes is not structural, but rather in regulating Rho activity via the GAP activity of MgcRacGAP or by its recruitment of the Rho GEF ECT-2 [Douglas and Mishima, 2010;Fabritius et al, 2011].…”

“…Myosin II is enriched in the meiotic contractile ring [Simerly et al, 1998;Maddox et al, 2005] and blebbistatin sensitivity of polar body cytokinesis in the clam Spisula demonstrates that actomyosin contractility closes the ring [Pielak et al, 2004]. Depletion of NMY-2 myosin II from C. elegans zygotes results in PBE failure [Shelton et al, 1999;Maddox et al, 2005], and depletion of the myosin phosphatase regulatory subunit deregulates the speed of ring closure [Fabritius et al, 2011]. Polar body production in mouse requires the Rho effector Rho-kinase [Elbaz et al, 2010], whose sole requirement in cytokinesis is thought to be myosin II light chain activation [Dean and Spudich, 2006].…”

“…Extrusion via polar relaxation requires a source of internal pressure. Global cortical stiffness is one candidate for this pressure [Fabritius et al, 2011]. Concomitant with the two rounds of PBE, echinoderm oocytes undergo periodic global increases in cortical stiffness, which are likely governed by cytoplasmic cell cycle regulatory machinery [reviewed by Rappaport, 1996] because they persist even in enucleated fragments [Yamamoto and Yoneda, 1983].…”