ZYG-9ch-TOG promotes the stability of acentrosomal poles via regulation of spindle microtubules in C. elegans oocyte 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. We exploited the auxin inducible degradation system to remove ZYG-9 from pre-formed spindles within minutes; this caused the poles to split apart and an unstable multipolar structure to form. 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 or TAC-1 surprisingly did not affect the assembly or stability of monopolar spindles, suggesting that these proteins are 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.

Polo-like kinase 1 prevents excess microtubule polymerization inC. elegansoocytes to ensure faithful meiosis

“…Microtubule growth has been linked to the amount of free tubulin in the cell (which should be the same in the oocyte regardless of PLK-1 AID) as well as the amount of ZYG-9 and TAC-1 (Srayko, Kaya, Stamford, & Hyman, 2005), which are associated with microtubule assembly in the C. elegans embryo (Bellanger & Gonczy, 2003). Since TAC-1 does not localize to the ectopic asters throughout the oocyte, we infer the same for ZYG-9 because these proteins co-localize in mitosis and meiosis (Cavin-Meza et al, 2022;Le Bot et al, 2003). Thus, we speculate that the asters throughout the oocyte may be due to altered activity of other microtubule regulatory factors.…”

“…Immunofluorescence was performed as described in Wolff et al 2022. Briefly, adult worms were picked into a 10 μL drop of Meiosis Medium (0.5 mg/mL Inulin, 25 mM HEPES, and 20% FBS in Leibovitz's L-15 Media (Gibco 11415-048)) (Laband, Lacroix, Edwards, Canman, & Dumont, 2018) on poly-L-lysine slides and dissected to remove oocytes.…”

The doublecortin-family kinase ZYG-8DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles

“…In recent years, C. elegans has emerged as a powerful model to dissect mechanisms that promote acentrosomal spindle assembly and maintenance, since rapid depletion methods have been developed that enable the removal of proteins from oocytes within minutes (Zhang et al, 2015; Divekar et al, 2021). These methods have made it possible to either deplete proteins prior to spindle formation, to probe for roles in spindle assembly, or to remove proteins from pre-formed spindles and then assess effects on the stability of the structure (Divekar et al, 2021; Cavin-Meza et al, 2022a; Cavin-Meza et al, 2022b).…”