Functions of Nonmuscle Myosin II in Assembly of the Cellular Contractile System

Abstract

The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system’s maturation heavily depends on nonmuscle myosin II (NMII). Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a load-dependent mechanism of NMII polymerization in association with attached bundles, leading to assembly of stress fibers and maturation of focal adhesions.

Characterization of Three Full-length Human Nonmuscle Myosin II Paralogs

“…Part of this is due to the crowded nature of the cytoskeleton, which makes it difficult to distinguish the small myosin filaments from the other elements, and part of this is due to difficulties in preserving their structures. Nevertheless, the images of filaments that are available bear a strong resemblance in size and shape to the in vitro filaments presented here (72,74,77). Of additional interest is the resemblance of the ribbon-like structures that we find at slightly lower ionic strength where the myosin filaments interact in both a head to head manner and in a parallel stacking arrangement.…”

“…In the cell, actomyosin networks are arranged in a variety of different geometries. These include sarcomeric structures such as those seen in stress fibers and purse strings as well as less ordered networks such as those seen at the peripheral region of the lamella (72)(73)(74). Because of the splaying and flexibility of individual NM II molecules from the filament, the actomyosin interactions seen here occur over a wide range of geometries, explaining how NM II filaments can maintain interactions with actin in the absence of well ordered sarcomere-like structures.…”

Myosin IIA is critical for organelle distribution and F-actin organization in megakaryocytes and platelets

“…17,18,37 In accordance with this hypothesis, we observed that the spatial organization of F-actin is abnormal in Myh9 2/2 MKs and actin polymerization is decreased in these platelets, indicating a role of myosin IIA in F-actin dynamics in MKs. The involvement of myosin II in actin polymerization/structuration has already been documented for F-actin cross-linking 38,39 or for catalyzing actin assembly at focal adhesions 39 or in dynamic structures where rapid actin filament turnover is required. [40][41][42] Thus, myosin IIA is likely to contribute to organelle distribution by organizing the actin meshwork necessary for organelle tethering and transport.…”

A novel interaction between Rab7b and actomyosin reveals a dual role in intracellular transport and cell migration

“…This difference in the effect of the two inhibitors on endosomal size might, however, be explained by the different mechanisms of action of the drugs because ML7 and Y27632, but not blebbistatin, interfere with phosphorylation of myosin II Shutova et al, 2012).…”