Methods for Investigating Cell Division Mechanisms in C. elegans

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

“…Given this initial validation, we created a Dynein AID strain that also expressed GFP::tubulin and GFP::histone and then assessed the effects of dynein depletion in oocytes by quantifying spindle morphology in intact worms ( Figure 1D , Table 1 ). This ‘germline counting’ method takes advantage of the fact that the C. elegans germline is arranged in a production-line fashion ( Wolff et al, 2022 ). Oocytes are fertilized in the –1 position of the germline, and then oocyte spindles assemble as the newly fertilized embryo moves through the spermatheca and into the +1 position ( Wolff et al, 2016 ).…”

“…Oocytes are fertilized in the –1 position of the germline, and then oocyte spindles assemble as the newly fertilized embryo moves through the spermatheca and into the +1 position ( Wolff et al, 2016 ). Under control conditions, bipolar oocyte spindles with focused poles are prevalent by the time the embryo reaches the +1 position ( Figure 1D ; Wolff et al, 2022 ). However, following 4 hr of auxin treatment, most spindles had splayed poles; a majority of spindles displayed microtubule bundles that did not come together at a single focused point on either side of the chromosomes (84/121, 69.4%), similar to what was observed following depletion of dynactin component DNC-1 in a previous study ( Crowder et al, 2015 ).…”

“…Our RNAi protocol is described in detail in Wolff et al, 2022 . Briefly, from an RNAi library ( Kamath et al, 2003 ), individual RNAi clones were picked and grown overnight at 37°C in LB with 100 μg/mL ampicillin.…”

“…Our IF protocol is described in detail in Wolff et al, 2022 . Briefly, adult worms, grown on either EV control RNAi or experimental RNAi, 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) ( Laband et al, 2018 ) in the center of a poly-L-lysine-coated glass slide.…”

“…Ethanol fixation and germline counting methods are described in more detail in Wolff et al, 2022 . Briefly, SMW46 worms were subjected to EV control RNAi (or klp-18(RNAi )), utilizing methods described above, and were grown to adulthood over 5 days.…”