In vitro contraction of cytokinetic ring depends on myosin II but not on actin dynamics

Abstract

Cytokinesis in many eukaryotes involves the contraction of an actomyosin-based contractile ring. However, the detailed mechanism of contractile ring contraction is not fully understood. Here, we establish an experimental system to study contraction of the ring to completion in vitro. We show that the contractile ring of permeabilized fission yeast cells undergoes rapid contraction in an ATP- and myosin-II-dependent manner in the absence of other cytoplasmic constituents. Surprisingly, neither actin polymerization nor its disassembly is required for contraction of the contractile ring, although addition of exogenous actin-crosslinking proteins blocks ring contraction. Using contractile rings generated from fission yeast cytokinesis mutants, we show that not all proteins required for assembly of the ring are required for its contraction in vitro. Our work provides the beginnings of the definition of a minimal contraction-competent cytokinetic ring apparatus.

The Cell Biology of Fission Yeast Septation

“…The close correlation between AMR closure and septum synthesis has encumbered the elucidation of whether the AMR is really pulling the plasma membrane enveloping the septum wall (94). In this regard, it has been traditionally thought that, as in metazoans, the S. pombe AMR exerts the pulling force needed to invaginate the plasma membrane to form the cleavage furrow (95,96). However, the frequent observation by electron microscopy of bent or misaligned growing septa in wild-type cells, together with the fact that AMR mutants are able to assemble septa, favored the hypothesis that the centripetal ingression of the growing septum wall would be able to push the septum plasma membrane (94).…”

Cytokinesis in Animal Cells

“…It is currently not know whether local accumulation of RhoA-GTP is sufficient to induce furrowing. Recent advances have paved the way for studying contractile ring action in a partially purified system in yeast (Mishra et al 2013;Stachowiak et al 2014). It will be exciting to define the biophysical basis for contractile ring dynamics and closure in animal cells.…”

Cytokinesis in Metazoa and Fungi

“…In the former case, ring constriction occurs in the presence of cytoplasmic extracts (Young et al 2010). In S. pombe, constriction occurs in permeabilized cells largely devoid of cytoplasmic components (Mishra et al 2013). During constriction of the ring, myosin II becomes progressively concentrated, whereas a significant portion of the filamentous actin disassembles.…”

“…The constriction rate is proportional to ring size, similar to that seen in C. elegans embryos (Carvalho et al 2009). During constriction, the rings slide along the inner leaflet of the droplet, rather than inducing deformations, as seen in permeabilized fission yeast and spheroplasts (Mishra et al 2013;Stachowiak et al 2014). Importantly, ring constriction under these conditions depends on motor activity-rigor conditions impair the assembly of rings seen in the absence of myosin.…”

“…Actin-filament-stabilizing drugs inhibit ring constriction; thus, constriction depends on actin filament disassembly. This approach has received renewed attention in budding and fission yeast (Young et al 2010;Mishra et al 2013). In the former case, ring constriction occurs in the presence of cytoplasmic extracts (Young et al 2010).…”