Nonmuscle Myosin II Isoforms Coassemble in Living Cells

Abstract

SUMMARY Non-muscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB and IIC), each of which possesses distinct biophysical properties and supports unique, as well as redundant, cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear if NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments, or if filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently-tagged versions of NM IIA, IIB and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms co-assemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly-spread cells, arguing for the existence of sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while co-assembled with other NM II isoforms.

Depletion of kinesin-12, a myosin-IIB interacting protein, promotes migration of cortical astrocytes

“…NMII plays core roles in many developmental and cellular processes, such as adhesion, migration, morphogenesis and cytokinesis, by pulling on actin filaments to generate contractile forces in cells. Recent studies have shown that individual NMII isoforms can perform both isoform-specific and isoform-redundant functions by assembling with other NMII isoforms (Beach et al, 2014), and have demonstrated the existence of NMII monomers, co-polymerization of NMIIA and NMIIB molecules, and contribution of both isoforms to early stages of contractile system assembly in cells (Shutova et al, 2014). These data show the potential of different NMII isoforms to assemble in different cellular localizations, related to different cytoskeleton functions.…”

The contractome – a systems view of actomyosin contractility in non-muscle cells

“…For example, NMIIA propels actin filaments the fastest whereas NMIIB can sustain tension for the longest period of time Wang et al, 2003). The isoforms can have distinct cellular localizations (Kolega, 1998;Betapudi et al, 2006;Smutny et al, 2010;Wang et al, 2011), although co-assembly of NMII isoforms also occurs in vivo (Beach et al, 2014;Shutova et al, 2014). However, because part of the contractome literature involves cells that have only a single representative of these proteins (e.g.…”

Spatial integration of E-cadherin adhesion, signalling and the epithelial cytoskeleton

“…Yet, their distinct dynamics [13], regulation and molecular composition indicates their separable identity: junctional actin contains -actin and myosin IIA, while -actin and myosin IIB are found at thin bundles [14,15]. As myosin IIA and myosin IIB can co-assemble on different types of actin filaments [16], it will be interesting to define how wide-spread the restricted distribution of these proteins among different epithelial cell types is.…”