Spherical spindle shape promotes perpendicular cortical orientation by preventing isometric cortical pulling on both spindle poles during C. elegans female meiosis

Abstract

Meiotic spindles are positioned perpendicular to the oocyte cortex to facilitate segregation of chromosomes into a large egg and a tiny polar body. In C. elegans, spindles are initially ellipsoid and parallel to the cortex before shortening to a near-spherical shape with flattened poles and then rotating to the perpendicular orientation by dyneindriven cortical pulling. The mechanistic connection between spindle shape and rotation has remained elusive. Here, we have used three different genetic backgrounds to manipulate spindle shape without eliminating dynein-dependent movement or dynein localization. Ellipsoid spindles with flattened or pointed poles became trapped in either a diagonal or a parallel orientation. Mathematical models that recapitulated the shape dependence of rotation indicated that the lower viscous drag experienced by spherical spindles prevented recapture of the cortex by astral microtubules emanating from the pole pivoting away from the cortex. In addition, maximizing contact between pole dynein and cortical dynein stabilizes flattened poles in a perpendicular orientation, and spindle rigidity prevents spindle bending that can lock both poles at the cortex. Spindle shape can thus promote perpendicular orientation by three distinct mechanisms.

Evidence for anaphase pulling forces duringC. elegansmeiosis

“…knl-1,3(kd) spindles frequently had two or more bivalents stacked end to end along the pole-to-pole axis of the spindle ( Fig. 3 A ) as previously described ( Vargas et al, 2019 ). Finally, knl-1,3(kd) spindles did not have apparent microtubule-free channels oriented in the pole-to-pole axis ( Fig.…”

“…Because Dumont et al (2010) found that GFP::KNL-3 remained in kinetochore cups after knl-1(RNAi) , we chose to double deplete KNL-1 and KNL-3 to more completely reduce kinetochore function during meiosis. Two previously described methods were used, auxin-induced degradation in strains with auxin-induced degrons (AIDs) appended to the endogenous knl-1 and knl-3 genes and GFP(RNAi) on strains with GFP appended to the endogenous knl-1 and knl-3 genes ( Vargas et al, 2019 ). After 48-h RNAi, GFP fluorescence was not observed around chromosomes in living embryos ( Fig.…”

“…Control spindles maintain a steady-state length of 8 µm for 7 min before initiating APC-dependent spindle shortening, followed by spindle rotation, followed by homolog separation as the spindle continues to shorten (anaphase A), and followed by a transition to spindle elongation (anaphase B; McNally et al, 2016 ; Yang et al, 2005 ). Because knl-1,3(kd) affects spindle dimensions, spindle rotation ( Vargas et al, 2019 ), chromosome congression, and anaphase movement, we used time relative to the initiation of spindle elongation to compare parameters between control and knl-1,3(kd) .…”

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

“…CLS-2 might promote oocyte contractile ring assembly and ingression more directly, through its regulation of microtubule stability during spindle assembly. Astral microtubules associated with this acentrosomal spindle are small and limited in number, but a recent study has proposed that they nevertheless can interact, through dynein, with cortical microtubules to engage pulling forces that move the spindle toward the cortex and rotate it from its initial parallel orientation relative to the oocyte cortex to a perpendicular orientation during polar body extrusion [ 60 ]. Astral microtubule interaction with the cortex might then also influence contractile ring assembly.…”

Caught in the Act: Live-Cell Imaging of Plant Meiosis

“…Key questions in meiotic research regarding the mechanism, function, and regulation of chromosome pairing, telomere bouquet formation, CO formation, and spindle formation have since then been assessed by live-cell imaging, which has provided new insights. These studies included further analyses in fission yeast (Tomita and Cooper, 2007), budding yeast (Conrad et al, 2008;Koszul and Kleckner, 2009;Lee et al, 2012Lee et al, , 2020, and Drosophila (Hughes et al, 2011;Colombié et al, 2013;Christophorou et al, 2015), as well as C. elegans (Vargas et al, 2019), and mammalian cells (Schuh and Ellenberg, 2007;Kitajima et al, 2011;Holubcová et al, 2013;Lee et al, 2015;Pfender et al, 2015;Kyogoku and Kitajima, 2017;Mogessie and Schuh, 2017;Enguita-Marruedo et al, 2018;Nikalayevich et al, 2018;Silva et al, 2020;Wang et al, 2020).…”