Microtubule assembly and pole coalescence: early steps in C. elegans oocyte meiosis I spindle assembly

Abstract

How oocytes assemble bipolar meiotic spindles in the absence of centrosomes as microtubule organizing centers remains poorly understood. We have used live cell imaging in Caenorhabditis elegans to investigate requirements for the nuclear lamina and for conserved regulators of microtubule dynamics during oocyte meiosis I spindle assembly, assessing these requirements with respect to recently identified spindle assembly steps. We show that the nuclear lamina is required for microtubule bundles to form a peripheral cage-like structure that appears shortly after oocyte nuclear envelope breakdown and surrounds the oocyte chromosomes, although bipolar spindles still assembled in its absence. Although two conserved regulators of microtubule nucleation, RAN-1 and γ-tubulin, are not required for bipolar spindle assembly, both contribute to normal levels of spindle-associated microtubules and spindle assembly dynamics. Finally, the XMAP215 ortholog ZYG-9 and the nearly identical minus-end directed kinesins KLP-15/16 are required for proper assembly of the early cage-like structure of microtubule bundles, and for early spindle pole foci to coalesce into a bipolar structure. Our results provide a framework for assigning molecular mechanisms to recently described steps in C. elegans oocyte meiosis I spindle assembly.

ZYG-9ch-TOGpromotes the stability of acentrosomal poles via regulation of spindle microtubules inC. elegansoocyte meiosis

“…In contrast, 50% of zyg-9(RNAi) embryos in the +1 position contained multipolar oocyte spindles while only 2% contained bipolar spindles, with the rest progressing to anaphase (Figure 1 – figure supplement 1D). These findings suggest that ZYG-9 is required for stable pole coalescence during acentrosomal spindle assembly, confirming the findings of a recent study (Chuang et al, 2020).…”

“…To confirm that degron::GFP:ZYG-9 retained its normal localization, we performed live imaging of oocytes and found that ZYG-9 begins to associate with microtubules at the multipolar stage, becoming enriched at the poles and increasing in intensity as the bipolar spindle forms (Figure 1 – figure supplement 2A, Figure 1B and Video 2), consistent with recent work (Chuang et al, 2020). Fixed imaging comparing the localization of degron::GFP::ZYG-9 to the spindle pole protein ASPM-1 confirmed these findings (Figure 1C, Figure 1 – figure supplement 2B).…”

“…Next, we sought to investigate how ZYG-9 promotes acentrosomal spindle stability. Since ZYG-9 localizes to spindle poles (Figure 1B, 1C, Figure 1 – figure supplement 2) and is required for both pole coalescence (Chuang et al, 2020) (Figure 1 – figure supplement 1, Figure 1D) and maintenance (Figure 2A-C, Figure 2 – figure supplement 1), one possibility is that a population of ZYG-9 at spindle poles functions to organize microtubule minus ends into a stable structure. However, since ZYG-9 depletion also causes microtubule bundles in the center of the spindle to splay (Figure 2B, 2D, Figure 2 – figure supplement 1) and decreases spindle length (Figure 2E), it is possible that the pole defects could instead be the result of a more global effect on spindle microtubules.…”

“…To date, the mechanisms of microtubule nucleation and polymerization during meiotic acentrosomal spindle assembly in C. elegans are not well characterized. Recent work in this system has implicated the nuclear lamina, the Ran GTPase gradient, and ɣ-tubulin in microtubule nucleation (Chuang et al, 2020). Additionally, the work presented in both Chuang et al .…”

“…Depletion of Msps results in multipolar spindles in Drosophila oocytes (Cullen and Ohkura, 2001). Moreover, in C. elegans , two early studies reported that spindles do not properly form following ZYG-9 depletion (Matthews et al, 1998; Yang et al, 2003), and a recent paper demonstrated that ZYG-9 is required for spindle pole coalescence (Chuang et al, 2020). However, whether ZYG-9 is required to maintain acentrosomal spindle stability once the bipolar spindle forms has not yet been investigated.…”