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

Abstract

During mitosis, centrosomes serve as microtubule organizing centers that guide the formation of a bipolar spindle. However, oocytes of many species lack centrosomes; how meiotic spindles establish and maintain these acentrosomal poles remains poorly understood. Here, we show that the microtubule polymerase ZYG-9ch-TOG is required to maintain acentrosomal pole integrity in C. elegans oocyte meiosis; following acute depletion of ZYG-9 from pre-formed spindles, the poles split apart and an unstable multipolar structure forms. Depletion of TAC-1, a protein known to interact with ZYG-9 in mitosis, caused loss of proper ZYG-9 localization and similar spindle phenotypes, further demonstrating that ZYG-9 is required for pole integrity. However, depletion of ZYG-9 surprisingly did not affect the assembly or stability of monopolar spindles, suggesting that ZYG-9 is not required for acentrosomal pole structure per se. Moreover, fluorescence recovery after photobleaching (FRAP) revealed that ZYG-9 turns over rapidly at acentrosomal poles, displaying similar turnover dynamics to tubulin itself, suggesting that ZYG-9 does not play a static structural role at poles. Together, these data support a global role for ZYG-9 in regulating the stability of bipolar spindles and demonstrate that the maintenance of acentrosomal poles requires factors beyond those acting to organize the pole structure itself.

C. elegans XMAP215/ZYG-9 and TACC/TAC-1 act at multiple times during oocyte meiotic spindle assembly and promote both spindle pole coalescence and stability

“…(F) RNAi knockdowns using previously reported conditions and endogenous GFP fusion strains [ 21 ]. As also reported by others [ 44 ], all GFP::TAC-1 signal is lost from spindle microtubules and poles after zyg-9 RNAi, while GFP::ZYG-9 is lost from poles but not spindle microtubules after tac-1(RNAi) . Scale bars = 5 μm.…”

“…(F) RNAi knockdowns using previously reported conditions and endogenous GFP fusion strains [21]. As also reported by others [44], all GFP::…”

“…Our results further suggest that ZYG-9 and TAC-1 act during metaphase to promote acentrosomal pole stability, and that this metaphase pole stability contributes to the maintenance of chromosome congression at the metaphase plate. Another recent study also indicates that ZYG-9 and TAC-1 are required for pole stability during oocyte meiosis I, and that ZYG-9 and TAC-1 are codependent each other for their localization to oocyte meiotic spindle poles [ 44 ].…”

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

“…Our results further suggest that ZYG-9 and TAC-1 act during metaphase to promote acentrosomal pole stability, and that this metaphase pole stability contributes to the maintenance of chromosome congression at the metaphase plate during oocyte meiotic cell division. Another recent study also reports data indicating that ZYG-9 and TAC-1 are required for pole stability during oocyte meiosis I, and in addition shows that ZYG-9 and TAC-1 are codependent each other for their localization to oocyte meiotic spindle poles (Mullen et al, 2022).…”

Multiple motors cooperate to establish and maintain acentrosomal spindle bipolarity in C. elegans oocyte meiosis

“…While dynein depletion splays spindle poles, microtubule bundles appear to remain stable and unperturbed, generating a rectangular spindle that can still progress through meiosis (albeit with spindle rotation defects). However, acute ZYG-9 depletion leads to a unstable multipolar spindle with defects in the middle of the spindle as well as at the poles, suggesting a broader role of ZYG-9 ( Mullen et al, 2022 ).…”