A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions.

No abstract

Actin cytoskeleton self-organization in two cell types, fibroblasts and epitheliocytes, was studied in cells confined to isotropic adhesive islands. In fibroblasts plated onto islands of optimal size, an initially circular actin pattern evolves into a radial pattern of actin bundles that undergo asymmetric chiral swirling before finally producing parallel linear stress fibers. Epitheliocytes, however, did not exhibit succession through all the actin patterns described above. Upon confinement, the actin cytoskeleton in non-keratinocyte epitheliocytes was arrested at the circular stage, while in keratinocytes it progressed as far as the radial pattern but still could not break symmetry. Epithelial–mesenchymal transition pushed actin cytoskeleton development from circular towards radial patterns but remained insufficient to cause chirality. Knockout of cytokeratins also did not promote actin chirality development in keratinocytes. Left–right asymmetric cytoskeleton swirling could, however, be induced in keratinocytes by treatment with small doses of the G-actin sequestering drug, latrunculin A in a transcription-independent manner. Both the nucleus and the cytokeratin network followed the induced chiral swirling. Development of chirality in keratinocytes was controlled by DIAPH1 (mDia1) and VASP, proteins involved in regulation of actin polymerization..

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.

The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It was selected in a chemical screen as a compound inhibiting formin-driven actin polymerization in vitro, but not polymerization of pure actin, and found to be active against several types of formins from different species (Rizvi et al., 2009). Here, in experiments with cultured fibroblasts, 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. While inhibition of myosin 2A in vitro required somewhat higher concentration of SMIFH2 than inhibition of retrograde flow and stress fiber contraction in cells, inhibition of several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5 by SMIFH2, was equally or more efficient than inhibition of formins. Since actin polymerization and myosin contractility are linked in many cytoskeleton processes, additional careful analysis is needed in each case when function of formins was proposed solely on the basis of experiment with SMIFH2.