Crosslinking activity of non-muscle myosin II is not sufficient for embryonic cytokinesis in C. elegans

Abstract

Cytokinesis in animal cells requires the assembly and constriction of a contractile actomyosin ring. Non-muscle myosin II is essential for cytokinesis, but the role of its motor activity remains unclear. Here, we examine cytokinesis in C. elegans embryos expressing non-muscle myosin motor mutants generated by genome editing. Two non-muscle motor-dead myosins capable of binding F-actin do not support cytokinesis in the one-cell embryo, and two partially motor-impaired myosins delay cytokinesis and render rings more sensitive to reduced myosin levels. Further analysis of myosin mutants suggests that it is myosin motor activity, and not the ability of myosin to crosslink F-actin, that drives the alignment and compaction of F-actin bundles during contractile ring assembly, and that myosin motor activity sets the pace of contractile ring constriction. We conclude that myosin motor activity is required at all stages of cytokinesis. Finally, characterization of the corresponding motor mutations in C. elegans major muscle myosin shows that motor activity is required for muscle contraction but is dispensable for F-actin organization in adult muscles..

Equatorial Non-muscle Myosin II and Plastin Cooperate to Align and Compact F-actin Bundles in the Cytokinetic Ring

“…F-actin flow analysis was performed using transgenic LifeAct::GFP, whose cortical levels are unaffected by myosin inhibition (Osorio et al, 2019). We chose LifeAct::GFP (Silva et al, 2016;Chan et al, 2019;Osorio et al, 2019) rather than LifeAct::mKate2 (Reymann et al, 2016), because in our hands one-cell embryos expressing LifeAct::mKate2 exhibited delayed furrow initiation and had a significantly reduced contractile ring constriction rate when compared to embryos expressing other fluorescent contractile ring components (Supplementary Figure S1A). In contrast, cytokinesis kinetics were normal in embryos expressing LifeAct::GFP.…”

“…(B) Stills of cortical images of an embryo expressing LifeAct::GFP at shallow deformation and furrow initiation with a schematic illustrating the transition from unidirectional cortical flows during ring assembly to bidirectional flows starting at the onset of ring constriction. (C) Perturbations used and their effect on the levels of endogenous and transgenic NMY-2 and LifeAct::GFP relative to controls with no RNAi, as described in Osorio et al (2019). (D) Kymographs of LifeAct::GFP cortical flows along the longitudinal axis in one-cell embryos for the conditions indicated in (C).…”

“…According to the latter, F-actin and myosin are directly recruited to the equatorial cortex from the cytoplasm, leading to local alignment of F-actin bundles (Kimura et al, 1996;Bement et al, 2005;Vavylonis et al, 2008;Uehara et al, 2010;Spira et al, 2017). The relative contribution of these two mechanisms to cytokinesis has been difficult to ascertain, because flows are myosin-dependent (Reymann et al, 2016) yet myosin perturbations may prevent cytokinesis simply because myosin-dependent force generation at the equator is essential for equatorial deformation and continued furrow ingression (Davies et al, 2014;Osorio et al, 2019). While actomyosin cortical flows towards the cell equator during cytokinesis have been described in several experimental systems, including C. elegans (Reymann et al, 2016;Khaliullin et al, 2018;Singh et al, 2019), they are less evident in some mammalian cells and are absent in yeast (Cao and Wang, 1990;Murthy and Wadsworth, 2005;Wu and Pollard, 2005;Vavylonis et al, 2008;Laplante et al, 2015).…”

“…We previously generated C. elegans expressing motor-dead mutants of myosin (NMY-2) and showed that myosin motor activity is essential for cytokinesis in the one-cell embryo (Osorio et al, 2019). In this study, we use these nmy-2 mutants, as well as a plst-1 mutant, to explore the role of cortical flows during cytokinesis in the one-cell C. elegans embryo.…”

Src-dependent NM2A tyrosine-phosphorylation regulates actomyosin dynamics

“…The expression of motor-dead NMY2 mutants in C. elegans impairs the development of embryos [75]. However, the motor activity of the NM2A Y158E is not affected in vitro, suggesting that the phenotypes attributed to NMY2 Y163E are independent from myosin mechanical activity.…”