High-Resolution Temporal Analysis Reveals a Functional Timeline for the Molecular Regulation of Cytokinesis

Abstract

Summary To take full advantage of fast-acting temperature-sensitive mutations, thermal control must be extremely rapid. We developed the Therminator, a device capable of shifting sample temperature in ~17s while simultaneously imaging cell division in vivo. Applying this technology to six key regulators of cytokinesis, we found that each has a distinct temporal requirement in the C. elegans zygote. Specifically, myosin-II is required throughout cytokinesis until contractile ring closure. In contrast, formin-mediated actin nucleation is only required during assembly and early contractile ring constriction. Centralspindlin is required to maintain division after ring closure, though its GAP activity is only required until just prior to closure. Finally, the Chromosomal Passenger Complex is required for cytokinesis only early in mitosis, but not during metaphase or cytokinesis. Together, our results provide a precise functional timeline for molecular regulators of cytokinesis using the Therminator, a powerful tool for ultra-rapid protein inactivation.

Progressive loss of RacGAP1/ ogre activity has sequential effects on cytokinesis and zebrafish development

“…However few of these studies have used live imaging analysis to characterize the cytokinesis defects in an intact embryo and to date the only animal where this has been done extensively is in C.elegans during the first cleavages. While studies in cultured cells and fixed Drosophila tissue depleted of RacGAP1 or MKLP1 homologs by RNAi or mutation indicate cells do not appear to form a cleavage furrow (Adams et al, 1998;D'Avino et al, 2006;Lekomtsev et al, 2012;Mishima et al, 2002;Somers and Saint, 2003;Sommi et al, 2010;Zavortink et al, 2005), live imaging of C. elegans eggs carrying equivalent mutations show that the first cleavage furrow forms correctly and ingresses extensively, but then regresses before a stable midbody is established (Canman et al, 2008;Davies et al, 2014;JantschPlunger et al, 2000;Powers et al, 1998;Raich et al, 1998). Our live imaging studies of cytokinesis in an intact vertebrate embryo carrying a presumptive null mutation show cytokinesis decays over time due to the elimination of maternal racgap1 transcripts.…”

A network of conserved formins, regulated by the guanine exchange factor EXC-5/FGD and the GTPase Cdc42, modulates tubulogenesis in vivo

“…To bypass the synthetic lethality of genetic combinations with cyk-1(0), we used a recessive, fast-acting and strong temperature-sensitive (ts) allele of cyk-1 (Davies et al, 2014;Jordan et al, 2016). We shifted cyk-1(ts) mutant embryos to the non-permissive temperature and maintained them at the restrictive temperature until they were L4 larvae or young adults, so that EC development occurred primarily or exclusively at the restrictive temperature (see Materials and Methods).…”

“…2B, C,D,F, animals were grown at 22 or 25°C and imaged by spinning disk confocal microscopy (see below). All strains in Figs 3 and 4 were analyzed using a temperature-shift strategy: adult hermaphrodites were allowed to lay eggs at 15°C for ∼24 h, then the adults were removed and F1 embryos were grown at 25°C, the restrictive temperature (Davies et al, 2014;Jordan et al, 2016), until L4 to young adult stage, when they were scored. We always examined the youngest animals in our temperature-shift experiments (i.e.…”

Meeting report – New York Symposium on Quantitative Biology of the Cell

“…In the C. elegans single-cell zygote, anterior-posterior polarity is established just after fertilization as different PAR proteins target to opposite sides of the cortex. Using a fluid device called 'Therminator' that allows rapid shifting of sample temperature (within 17 s) while simultaneously imaging cell division at high spatiotemporal resolutions (Bioptechs; Davies et al, 2014), Canman and colleagues made an unexpected finding when they mapped the precise time window required for formin-and/or myosin-II based cytokinesis. At semipermissive temperatures, which support successful cytokinesis in temperature-sensitive formin and myosin-II single mutants, a temperature-sensitive formin and myosin-II double mutant displayed a synthetic cytokinesis failure defect.…”