Microtubules Oppose Cortical Actomyosin-driven Membrane Ingression During C. Elegans Meiosis I Polar Body Extrusion

Abstract

During C. elegans oocyte meiosis I cytokinesis and polar body extrusion, cortical actomyosin is locally remodeled to assemble a contractile ring that forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness limits membrane ingression throughout the oocyte during meiosis I polar body extrusion. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a group of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize during meiosis I to structures called linear elements, which are present within the assembling oocyte spindle and also are distributed throughout the oocyte in proximity to, but appearing to underlie, the actomyosin cortex. 

Introduction

Animal cell shape and morphogenesis are influenced by both tension and elasticity within the cell cortex [1–4]. Cortical tension and elasticity both depend on the actomyosin cytoskeleton and its associated proteins. Non-muscle myosin and microfilament architecture are largely responsible for generating tension [5], while increased cross-linking of cortical microfilaments to the plasma membrane by Ezrin/Radixin/Moesin (ERM) proteins during the cell rounding associated with mitosis generates decreased elasticity and hence increased cortical stiffness [6–8]. While there is growing evidence for crosstalk between the microtubule and microfilament cytoskeletons [9,10], microtubules are generally viewed as forming distinct cytoskeletal structures that are not part of the animal cell cortex.

Materials and methods

Feeding RNAi Knockdown

All RNAi experiments were carried out by feeding E. coli strain HT115(DE3) induced by IPTG to express double-stranded RNA corresponding to the following genes: bub-1, cls-2, klp-7, knl-1, rod-1 and zyg-9. First bacteria clones were picked from an RNAi library and grown on LB Agar plate with Ampicillin [63]. Each bacterial clone was miniprepped using the Qiagen kit for subsequent sequence confirmation. Hypochlorite synchronized L1 larvae were grown on standard nematode growth medium plates, washed with M9 three times and then plated on the induced RNAi plates and grown at 20°C until imaging. The feeding times were chosen such that if treatment were extended for an additional 6 more hours, 90% or more of the adult worms became sterile. For bub-1, cls-2, klp-7, rod-1 and zyg-9 worms were fed for 48–52 hours. For knl-1 RNAi, L1 larvae were grown until L4 stage and placed onto knl-1 RNAi plates for 35–40 hours. For auxin induced degron (AID) experiments, feeding times were chosen until hatching rate reached 0%. For all AID strains, hypochlorite synchronized L1 larvae were grown on standard nematode growth medium plates (NGM) until L4 stage, where worms were transferred onto NGM with 1mM Auxin plates. For all strains, worms were placed onto the 1mM Auxin plates for 13 hours before imaging.

Results

KNL-1, BUB-1 and CLS-2 co-localize to linear elements that underlie the cortex during meiosis I
To determine when KNL-1, BUB-1 and CLS-2 are present, and if they co-localize to linear elements, we used spinning disk confocal microscopy and live cell imaging to track fluorescent protein fusions to each protein (see Materials and Methods). First, we examined their localization throughout meiosis I and II, imaging in utero oocytes that express GFP fused to one of the three kinetochore proteins, and mCherry fused to a histone (mCherry::H2B) to mark chromosomes. As reported previously [11,15,16], we detected the GFP fusions to KNL-1, BUB-1 and CLS-2 shortly after nuclear envelope breakdown during meiosis I, in association with chromosomes and also in linear elements enriched within the assembling spindle and present less densely throughout the oocyte (Figs 2A and S1 and S1 Movie). The KNL-1, BUB-1, and CLS-2 linear elements persisted until the beginning of anaphase B, when they became undetectable, approximately 13 minutes after nuclear envelope breakdown and just as polar body extrusion began. 

Discussion

We have shown that two subunits of an outer kinetochore sub-complex—the kinase BUB-1 and the CLASP family member CLS-2—along with the outer kinetochore scaffolding protein KNL-1, co-localize both to linear elements associated with the oocyte meiosis I spindle, and to related sub-cortical patches that are mobile and distributed throughout the oocyte, underlying its cortex until they dissipate at the beginning of anaphase B during C. elegans meiosis I cell division. As previously documented at kinetochores, CLS-2 localization to these sub-cortical patches required BUB-1 and KNL-1. However, unlike at kinetochores, we also observed extensive mutual dependence for sub-cortical patch localization. This analysis relied on partial depletions of these proteins, as KNL-1 and BUB-1 are both required for fertility and cannot be analyzed in null oocyte backgrounds. Thus, our results may not fully reveal either the dependencies of these proteins on each other either for patch localization, or their other requirements. Nevertheless, knocking down any one of these sub-cortical patch components resulted both in an altered distribution of sub-cortical microtubules and in excess membrane ingression throughout the oocyte during polar body extrusion.

Acknowledgments

We thank Arshad Desai, Julien Dumont, Reto Gassmann, Frank McNally, and the Caenorhabditis Genetics Center (funded by the NIH Office of Research Infrastructure Programs) for C. elegans strains, Dan Dickinson and Alexander Cartagena-Rivera for helpful discussions, and Chris Doe for sharing equipment.

Citation: Quiogue AR, Sumiyoshi E, Fries A, Chuang C-H, Bowerman B (2023) Microtubules oppose cortical actomyosin-driven membrane ingression during C. elegans meiosis I polar body extrusion. PLoS Genet 19(10): e1010984. https://doi.org/10.1371/journal.pgen.1010984

Editor: Monica P. Colaiácovo, Harvard Medical School, UNITED STATES

Received: May 22, 2023; Accepted: September 19, 2023; Published: October 2, 2023

Copyright: © 2023 Quiogue et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was funded by NIH grant R35 GM131749 (A.R.Q., E.S., C.-H.C., B.B.), NIH Training Grant T32HD007348-32 (A.R.Q.), and the University of Oregon Office of the Vice-President for Research (A.F.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010984#abstract0