Abstract
ABSTRACTThe small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formin from different species. Here, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro, and found that SMIFH2 inhibits activity of myosin ATPase and the ability to translocate actin filaments in the gliding actin in vitro motility assay. Inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 compared with that needed to inhibit retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, including bovine myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficiently than it inhibits formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions.This article has an associated First Person interview with Yukako Nishimura, joint first author of the paper.
Highlights
Formins are a large and diverse class of actin associated proteins that are evolutionarily conserved in nature (Breitsprecher and Goode, 2013; Schönichen and Geyer, 2010; van Gisbergen and Bezanilla, 2013)
The initial observation which triggered this study was inhibition of traction forces exerted by REF52 fibroblast upon treatement with 30 μM of SMIFH2
We further investigated how SMIFH2 would affect the ATP-dependent contractility of linear ventral stress fibers in REF52 cells
Summary
Formins are a large and diverse class of actin associated proteins that are evolutionarily conserved in nature (Breitsprecher and Goode, 2013; Schönichen and Geyer, 2010; van Gisbergen and Bezanilla, 2013). Formin activities include nucleation and processive elongation of actin filaments (Courtemanche, 2018; Paul and Pollard, 2009; Zigmond, 2004); some formins bundle actin filaments (Harris et al, 2006; Michelot et al, 2006; Schönichen et al, 2013) and bind to microtubules (Bartolini et al, 2008; Chesarone et al, 2010; Gaillard et al, 2011). The FH2 domains form dimers, which can nucleate actin filaments and function as processive caps at the filament plus (barbed) ends (Aydin et al, 2018; Courtemanche, 2018; Goode and Eck, 2007; Paul and Pollard, 2009). Combined action of FH1 and FH2 domains strongly accelerates filament growth
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