The formin inhibitor SMIFH2 inhibits members of the myosin superfamily

Abstract

The 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 formins from different species (Rizvi et al., 2009). 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 myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. The inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 than for the inhibition of retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficient than inhibition of formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions.

Crosstalk between myosin II and formin functions in the regulation of force generation and actomyosin dynamics in stress fibers

“…5G), or measured by pillar deflection (Supplementary Figure 2B). Of note, the concentration of SMIFH2 used in these experiments ( 30µM) was lower than the concentration of this drug that inhibits myosin II A [27].…”

“…Complete suppression of photoconverted actin incorporation was observed also upon treatment with 30µM of SMIFH2, (Fig. 3) which is known to interfere with formin family protein interaction with actin [36] as well as with activity of several types of myosins [27] . However, treatment with a specific inhibitor of Arp2/3 dependent actin polymerization, CK666 [37,38] did not affect incorporation of the photoconverted mEos3.2-b-actin into the stress fibers even at 100 µM concentration (Fig.…”

“…We used latrunculin A, jasplakinolide, CK666, and SMIFH2 compounds characterized above. As mentioned previously, in addition to formin inhibition, SMIFH2 can inhibit myosin family proteins including non-muscle myosin IIA [27]. In this study, we carefully compared the effects of SMIFH2 and myosin II inhibitors (pAB and Y27632) on traction force generation to dissect out the two activities of this drug and reveal the role of formins in the regulation of cell contractility.…”

“…In this section, we summarize the methodological approaches used to assess actomyosin cytoskeleton dynamics and the cell generated traction forces. First, we used photoconvertible bactin (mEos3.2-b-actin) from Addgene/Michael Davidson lab for assessment of actin incorporation in the ventral stress fibres and focal adhesions [27]. In this set up, we illuminated a circular region (6.5 µm diameter) in the central part of the cell expressing mEos3.2-actin for three seconds to induce green to red photoconversion of labelled actin.…”

“…These data demonstrate that inhibition of traction forces by SMIFH2 can be prevented by increase of actin filament cross-linking by a-actinin. The difference between SMIFH2 and myosin II inhibition strongly suggests that the loss of tension cannot be attributed to an off-target effect of SMIFH2 on myosin II activity [27], and hence that formins are directly involved in the maintenance of traction forces.…”