Assembly Of myosin-IIA Filaments Research Articles

Forces and constraints controlling podosome assembly and disassembly

Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors. First, we describe the process of podosome disassembly induced by activation of myosin-IIA filament assembly. Next, we find that podosome integrity and dynamics depends upon membrane tension and can be experimentally perturbed by osmotic swelling and deoxycholate treatment. We have also found that podosomes can be disrupted in a reversible manner by single or cyclic radial stretching of the substratum. We show that disruption of podosomes induced by osmotic swelling is independent of myosin-II filaments. The inhibition of the membrane sculpting protein, dynamin-II, but not clathrin, resulted in activation of myosin-IIA filament formation and disruption of podosomes. The effect of dynamin-II inhibition on podosomes was, however, independent of myosin-II filaments. Moreover, formation of organized arrays of podosomes in response to microtopographic cues (the ridges with triangular profile) was not accompanied by reorganization of myosin-II filaments. Thus, mechanical elements such as myosin-II filaments and factors affecting membrane tension/sculpting independently modulate podosome formation and dynamics, underlying a versatile response of these adhesion structures to intracellular and extracellular cues. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

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

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes. Myosin IIA filaments are indispensable effectors in the microtubule-driven regulation of integrin-mediated adhesions. Myosin IIA filament assembly depends on Rho activation by the RhoGEF GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.

Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO.

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly.

Gelsolin and Non-muscle Myosin IIA Interact to Mediate Calcium-regulated Collagen Phagocytosis

The formation of adhesion complexes is the rate-limiting step for collagen phagocytosis by fibroblasts, but the role of Ca(2+) and the potential interactions of actin-binding proteins in regulating collagen phagocytosis are not well defined. We found that the binding of collagen beads to fibroblasts was temporally and spatially associated with actin assembly at nascent phagosomes, which was absent in gelsolin null cells. Analysis of tryptic digests isolated from gelsolin immunoprecipitates indicated that non-muscle (NM) myosin IIA may bind to gelsolin. Immunostaining and immunoprecipitation showed that gelsolin and NM myosin IIA associated at collagen adhesion sites. Gelsolin and NM myosin IIA were both required for collagen binding and internalization. Collagen binding to cells initiated a prolonged increase of [Ca(2+)](i), which was absent in cells null for gelsolin or NM myosin IIA. Collagen bead-induced increases of [Ca(2+)](i) were associated with phosphorylation of the myosin light chain, which was dependent on gelsolin. NM myosin IIA filament assembly, which was dependent on myosin light chain phosphorylation and increased [Ca(2+)](i), also required gelsolin. Ionomycin-induced increases of [Ca(2+)](i) overcame the block of myosin filament assembly in gelsolin null cells. We conclude that gelsolin and NM myosin IIA interact at collagen adhesion sites to enable NM myosin IIA filament assembly and localized, Ca(2+)-dependent remodeling of actin at the nascent phagosome and that these steps are required for collagen phagocytosis.

Regulation of Nonmuscle Myosin-IIA Filament Assembly/Disassembly

The assembly of myosin-II into filaments is required for cytokinesis, cell motility, and the maintenance of cell morphology. Myosin-II filament dynamics are under strict spatial and temporal control; however, the molecular mechanisms modulating assembly and disassembly are poorly understood. Our studies focus on the contribution of the C-terminal coiled-coil and the nonhelical tailpiece to the regulation of myosin-IIA filament assembly. We probed the intrinsic dynamics of the coiled-coil and its interaction with S100A4, a major metastasis factor. Using sedimentation equilibrium, hydrogen-deuterium exchange, and thermal melt CD spectroscopy, we showed that C-terminal coiled-coil of the myosin-IIA heavy chain exhibits significant conformational plasticity. We propose a mechanism in which S100A4 binding to the C-terminal coiled-coil locally unzips the two polypeptide chains of the dimeric myosin-IIA coiled-coil, and thus modulates myosin-IIA filament assembly. We are testing this hypothesis by using electron spin resonance (EPR) spectroscopy to monitor conformational changes in the myosin-IIA coiled-coil upon S100A4 binding.

Regulation of Nonmuscle Myosin IIA Assembly

In eukaryotic cells, nonmuscle myosin II filament assembly is critical for essential motile processes such as cytokinesis, cell motility, and the maintenance of cell morphology. Although myosin II filament dynamics are believed to be under strict spatial and temporal control, the mechanisms modulating filament assembly and disassembly are poorly understood. In this work, we examined the molecular mechanisms regulating myosin IIA filament assembly that rely on the intrinsic dynamics of the C-terminal coiled-coil of the myosin IIA heavy chain and on non-covalent interactions with S100A4 protein, a major metastasis factor. Sedimentation equilibrium, hydrogen-deuterium exchange, and thermal melt CD spectroscopy showed that the C-terminal coiled-coil of the non-muscle myosin IIA heavy chain exhibits significant conformational plasticity. Based on these observations, we propose that the plasticity of the C-terminal coiled-coil is an inherent regulatory mechanism for modulating myosin IIA filament assembly. We are testing this hypothesis by introducing stabilizing mutations into the C-terminal coiled-coil and examining how the loss of plasticity modulates salt-dependent oligomerization of myosin IIA and filament assembly as assayed by turbidity and critical concentration measurements.

Mts1 regulates the assembly of nonmuscle myosin-IIA.

The formation of myosin-II filaments is fundamental to contractile and motile processes in nonmuscle cells, and elucidating the mechanisms controlling filament assembly is essential for understanding how myosin-II rapidly responds to changing conditions within the cell. Several proteins including KRP and a novel 38 kDa protein (1, 2) have been shown to modulate filament assembly through the stabilization of myosin-II assemblies. In contrast, we demonstrate that mts1, a member of the Ca(2+)-regulated S100 family of proteins, may regulate the monomeric, unassembled state in an isoform-specific manner. Biochemical analyses demonstrate that mts1 has a 9-fold higher affinity for myosin-IIA filaments than for myosin-IIB filaments. At stoichiometric levels, mts1 inhibits the assembly of myosin-IIA monomers into filaments and promotes the disassembly of myosin-IIA filaments into monomers; however, mts1 has little effect on the assembly properties of myosin-IIB. Using a solution based-assay, we have demonstrated that mts1 binds to residues 1909-1924 of the myosin-IIA heavy chain, which is near the C-terminal tip of the alpha-helical coiled-coil. The observation that mts1 binds a linear sequence of approximately 16 amino acids is consistent with other S100 family members, which bind linear sequences of 13-22 residues in their protein targets. In addition, mts1 increases the critical monomer concentration for myosin-IIA filament assembly by approximately 11-fold. Kinetic assembly assays indicate that the elongation rate and the extent of polymerization depend on the initial myosin-IIA concentration; however, mts1 had only a small affect on the half-time for assembly and predominately affected the extent of myosin IIA polymerization. Altogether, these observations are consistent with mts1 regulating myosin IIA assembly by monomer sequestration and suggest that mts1 regulates cell shape and motility through the modulation of myosin-IIA function.