Motor Activity Dependent and Independent Functions of Myosin II Contribute to Actomyosin Ring Assembly and Contraction in Schizosaccharomyces pombe

Abstract

SummaryCytokinesis depends on a contractile actomyosin ring in many eukaryotes [1, 2, 3]. Myosin II is a key component of the actomyosin ring, although whether it functions as a motor or as an actin cross-linker to exert its essential role is disputed [1, 4, 5]. In Schizosaccharomyces pombe, the myo2-E1 mutation affects the upper 50 kDa sub-domain of the myosin II heavy chain, and cells carrying this lethal mutation are defective in actomyosin ring assembly at the non-permissive temperature [6, 7]. myo2-E1 also affects actomyosin ring contraction when rings isolated from permissive temperature-grown cells are incubated with ATP [8]. Here we report isolation of a compensatory suppressor mutation in the lower 50 kDa sub-domain (myo2-E1-Sup1) that reverses the inability of myo2-E1 to form colonies at the restrictive temperature. myo2-E1-Sup1 is capable of assembling normal actomyosin rings, although rings isolated from myo2-E1-Sup1 are defective in ATP-dependent contraction in vitro. Furthermore, the product of myo2-E1-Sup1 does not translocate actin filaments in motility assays in vitro. Superimposition of myo2-E1 and myo2-E1-Sup1 on available rigor and blebbistatin-bound myosin II structures suggests that myo2-E1-Sup1 may represent a novel actin translocation-defective allele. Actomyosin ring contraction and viability of myo2-E1-Sup1 cells depend on the late cytokinetic S. pombe myosin II isoform, Myp2p, a non-essential protein that is normally dispensable for actomyosin ring assembly and contraction. Our work reveals that Myo2p may function in two different and essential modes during cytokinesis: a motor activity-independent form that can promote actomyosin ring assembly and a motor activity-dependent form that supports ring contraction.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring

“…We find Spn3 near the membrane, consistent with its assembly into membrane-bound oligomers with additional septin subunits ( An et al, 2004 ). In addition to membrane-binding proteins, the C-terminal tail of the essential contractile ring myosin-II Myo2 ( Kitayama et al, 1997 ; Palani et al, 2017 ) is near the membrane, as was observed in cytokinesis precursor nodes ( Laporte et al, 2011 ; Laplante et al, 2016 ). The N-terminus of the formin Cdc12 is also found near the membrane, in accord with direct binding to the F-BAR domain of Cdc15 ( Willet et al, 2015b ; Carnahan and Gould, 2003 ).…”

“…At similar intermediate distances from the membrane, we find the tail domains of two additional myosins that contribute to S. pombe cytokinesis: the non-essential myosin-II, Myp2 ( Bezanilla et al, 1997 ; Mulvihill and Hyams, 2003 ), as well as a myosin-V, Myo51 ( Win et al, 2001 ; Tang et al, 2016 ). Though both are non-essential, Myp2 is implicated in ring constriction ( Palani et al, 2017 ; Laplante et al, 2015 ) while Myo51 contributes to ring formation and constriction ( Tang et al, 2016 ).…”

Crosslinkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis

“…In addition, cell biological work with budding yeast and theoretical modeling demonstrated that ring closure can be powered by the thermal ratcheting of non-motor 3 crosslinkers along depolymerizing F-actin (Mendes Pinto et al, 2012;Oelz et al, 2015). Similarly, in fission yeast, a motor-impaired NMM-II mutant that robustly crosslinks F-actin is sufficient for ring assembly (Palani et al, 2017). Collectively, these studies suggest that F-actin crosslinking promotes cytoskeletal remodeling during cytokinesis, and ring closure in animal and fungal cells.…”

Molecular form and function of the cytokinetic ring

“…Intermediate layer: accessory protein network individual CR components, particularly those required for CR formation, contribute to contraction (Mishra et al, 2013;Huang et al, 2016a;Chew et al, 2017;Palani et al, 2017). These studies defined the minimal requirements for CR contraction in this system as the presence of F-actin, myosin-II ATPase activity and a balance of actin-crosslinking proteins (Mishra et al, 2013); surprisingly, the actin severer cofilin is not essential.…”