Self organization of the actomyosin cytoskeleton in fibroblasts

We collaborated with Sasha Bershadsky on a project utilising structured illumination microscopy to follow the dynamics of non-muscle myosin IIA in rat embryonic fibroblasts (REF52) cells. We first characterised the structure of stress fibers, resolving the repetitive sarcomere-like unit (see panel G in figure below). Next, we followed the co-assembly of myosin filaments with actin filaments into stress fibers and their further alignment into “myosin stacks”, which are supracellular structures that run perpendicular to the direction of the stress fibers. Various chemical inhibitors and siRNA knockdown were used to demonstrate that the assembly of myosin stacks depends on actin assembly/disassembly dynamics and myosin motor activity. This work was published in Nature Cell Biology 19(2):133-141 doi: 10.1038/ncb3466.

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We are now further studying the proteins and molecular mechanisms involved in myosin IIA stack formation. An siRNA screen of candidate Contractome proteins has led to the discovery of two opposite phenotypes, as shown below: 1. collapse of stress fibers into large bundles that completely loose the order in myosin organization and 2. dramatic enhancement of myosin stack formation, causing the fibroblast to look like muscle cells. We are currently studying the genes that give rise to these strikingly opposing phenotypes.

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