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Non-muscle myosin 2 (NM2) motors are the major contractile machines in most cell types. Unsurprisingly, these ubiquitously expressed actin-based motors power a plethora of subcellular, cellular and multicellular processes. In this Cell Science at a Glance article and the accompanying poster, we review the biochemical properties and mechanisms of regulation of this myosin. We highlight the central role of NM2 in multiple fundamental cellular processes, which include cell migration, cytokinesis, epithelial barrier function and tissue morphogenesis. In addition, we highlight recent studies using advanced imaging technologies that have revealed aspects of NM2 assembly hitherto inaccessible. This article will hopefully appeal to both cytoskeletal enthusiasts and investigators from outside the cytoskeleton field who have interests in one of the many basic cellular processes requiring actomyosin force production.

Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions, including endocytosis and organelle transport pathways. Cell type–specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the glycosylphosphatidylinositol-anchored protein uromodulin (UMOD) and gradual loss of Na+ K+ 2Cl– cotransporter from the apical membrane of the thick ascending limb epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules and activation of ER stress and unfolded protein response pathways in Myh9&10-cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress–mediated progressive renal tubulointerstitial disease. These observations establish cell type–specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin., Adult mouse renal epithelium specific knockout of Myh9 and Myh10 genes result in kidney disease.

The peri-centrosomal localization and morphology of the Golgi apparatus depends largely on the microtubule cytoskeleton and the microtubule motor protein dynein. Recent studies proposed that myosin 18Aα (M18Aα) also contributes to Golgi morphology by binding the Golgi protein GOLPH3 and walking along adjacent actin filaments to stretch the Golgi into its classic ribbon structure. Biochemical analyses have shown, however, that M18A is not an actin-activated ATPase and lacks motor activity. Our goal, therefore, was to define the precise molecular mechanism by which M18Aα determines Golgi morphology. We show that purified M18Aα remains inactive in the presence of GOLPH3, arguing against the Golgi-specific activation of the myosin. Using M18A-specific antibodies and expression of GFP-tagged M18Aα, we find no evidence that it localizes to the Golgi. Moreover, several cell lines with reduced or eliminated M18Aα expression exhibited normal Golgi morphology. Interestingly, actin filament disassembly resulted in a marked reduction in lateral stretching of the Golgi in both control and M18Aα-deficient cells. Importantly, this reduction was accompanied by an expansion of the Golgi in the vertical direction, vertical movement of the centrosome, and increases in the height of both the nucleus and the cell. Collectively, our data indicate that M18Aα does not localize to the Golgi or play a significant role in determining its morphology, and suggest that global F-actin disassembly alters Golgi morphology indirectly by altering cell shape. This article is protected by copyright. All rights reserved.

The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

Murugesan et al. report that actomyosin arcs at the T cell synapse are formin-generated structures that directly propel T cell receptor cluster movement. The authors reveal the origin, organization, and functions of a major cytoskeletal network during synapse maturation., Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T–B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.

Non-muscle myosin IIB plays a major role in applying force on the nucleus to facilitate nuclear translocation through tight spaces during 3D invasive migration, while non-muscle myosin IIA is critical for generating force during active protrusion., Non-muscle myosin II (NMII) is reported to play multiple roles during cell migration and invasion. However, the exact biophysical roles of different NMII isoforms during these processes remain poorly understood. We analyzed the contributions of NMIIA and NMIIB in three-dimensional (3D) migration and in generating the forces required for efficient invasion by mammary gland carcinoma cells. Using traction force microscopy and microfluidic invasion devices, we demonstrated that NMIIA is critical for generating force during active protrusion, and NMIIB plays a major role in applying force on the nucleus to facilitate nuclear translocation through tight spaces. We further demonstrate that the nuclear membrane protein nesprin-2 is a possible linker coupling NMIIB-based force generation to nuclear translocation. Together, these data reveal a central biophysical role for NMIIB in nuclear translocation during 3D invasive migration, a result with relevance not only to cancer metastasis but for 3D migration in other settings such as embryonic cell migration and wound healing.

Janelia Farm Research Campus, Howard Hughes MedicalInstitute, Ashburn, VA 20147, USASummaryNonmuscle myosin II (NM II) powers myriad developmentaland cellular processes, including embryogenesis, cellmigration, and cytokinesis [1]. To exert its functions, mono-mers of NM II assemble into bipolar filaments that produce acontractile force on the actin cytoskeleton. Mammalian cellsexpress up to three isoforms of NM II (NM IIA, IIB, and IIC),each of which possesses distinct biophysical propertiesand supports unique as well as redundant cellular functions[2–8]. Despite previous efforts [9–13], it remains unclearwhether NM II isoforms assemble in living cells to producemixed (heterotypic) bipolar filaments or whether filamentsconsist entirely of a single isoform (homotypic). We ad-dressed this question using fluorescently tagged versionsof NM IIA, IIB, and IIC, isoform-specific immunostaining oftheendogenousproteins,andtwo-color totalinternalreflec-tion fluorescence structured-illumination microscopy, orTIRF-SIM, to visualize individual myosin II bipolar filamentsinside cells. We show that NM II isoforms coassemble intoheterotypic filaments in a variety of settings, includingvarious types of stress fibers, individual filamentsthroughout the cell, and the contractile ring. We also showthatthedifferentialdistributionofNMIIAandNMIIBtypicallyseen in confocal micrographs of well-polarized cells is re-flected in the composition of individual bipolar filaments.Interestingly, this differential distribution is less pro-nounced in freshly spread cells, arguing for the existenceof a sorting mechanism acting over time. Together, ourwork argues that individual NM II isoforms are potentiallyperforming both isoform-specific and isoform-redundantfunctions while coassembled with other NM II isoforms.Results and DiscussionTIRF-SIM Reveals Individual NM II Bipolar FilamentsIn an initial effort to identify individual nonmuscle myosin II(NMII)bipolarfilaments,weexpressedNMIIAwithanN-termi-nalEGFPtag(EGFP-NMIIA;notethatalltagswerefusedtotheNM II heavy chain; Figure 1A) in U2OS cells and imaged thecells by total internal reflection fluorescence structured-illumi-nation microscopy (TIRF-SIM) (Figure 1C), a TIRF version of apreviously established SIM technique [14] that provides two-color imaging capability and achieves a lateral resolution ofw100 nm. This resolution should allow unequivocal identifica-tionofw300nmNMIIbipolarfilaments[15,16],unlikeimagingperformed by conventional microscopy, where the lateralresolution is w250 nm (although see [17]). In TIRF-accessibleregions of the lamellar extensions and the cell interior, imagesrevealed what appeared to be individual NM IIA filamentspossessing two puncta spaced w300 nm apart (Figure 1Cand insets C1 and C2). These putative filaments were usuallyembedded in actin networks (Figure 1D and insets D1 andD2) or aligned with linear actin filaments/bundles (Figure 1EandinsetsE1andE2)labeledwithF-tractin,anF-actinreporter[18, 19]. In regions rich in transverse arcs and ventral stress fi-bers, however, EGFP-NM IIA puncta were too numerous andclose together to identify individual filaments unequivocally(Figure1CandinsetC3).Toconfirmthatthese300nm-spacedpuncta correspond to individual NM IIA bipolar filaments andto resolve these structures in filament-rich regions of the cell,weusedNMIIAwithaC-terminalmAppletag(NMIIA-mApple;Figure 1A). Because NM II filaments are bipolar, coexpressionof NM IIA-mApple with EGFP-NM IIA should result in filamentswith EGFP puncta at both ends of the filament (correspondingto the N termini of the head domains) bifurcated by a singlemApple punctum (corresponding to C termini of the taildomains)(Figure1B).Consistently,whenthesetwoconstructswere coexpressed in U2OS cells, we observed two greenpuncta w300 nm apart that were bifurcated by a single redpunctum (Figure 1F and inset F1). Moreover, these two-colorstructures were readily resolvable in filament-rich regions ofthecell(Figure1FandinsetsF2andF3).Importantly,thelocal-izationofC-terminallytaggedNMIIAwasqualitativelyindistin-guishable from N-terminally tagged NM IIA, arguing that theC-terminal tag has no obvious deleterious effects on bipolarfilament structure (although minor effects cannot be entirelyruledout).WenotethatasimilarapproachemployinganN-ter-minalantibodyandaC-terminalfluorophorewasusedrecentlyto identify bipolar filaments containing NM IIC in epithelia [17].Exogenous NM II Isoforms Form Heterotypic FilamentsTo determine whether NM II isoforms form heterotypicfilaments, we coexpressed NM IIA-mApple and EGFP-NMIIB in U2OS cells. Although both isoforms are overexpressed,the ratio of NM IIA to NM IIB in transfected cells remainsessentiallythesameasinuntransfectedcells(w25:1)becausethe fold increase over endogenous protein levels is approxi-mately the same for both isoforms (w2.5- to 3-fold) (see Fig-ure S1 and Table S1 available online). Confocal microscopydemonstrated that these two isoforms ‘‘colocalize’’ to somedegree throughout most of the cell (Figure 2A), although NMIIAis enrichedinperipherallamellarelative toNMIIB,whereasNM IIB is enriched in central and posterior regions relative toNM IIA, as reported by others [8, 20, 21] (see also below). Todetermine whether this ‘‘colocalization’’ corresponds at leastin part to heterotypic filaments of NM IIA and NM IIB, weimaged using TIRF-SIM. Figure 2B shows that the green-red-green signal expected for heterotypic filaments is indeedvery common in TIRF-accessible interior regions of cells (seealso insets B1 and B2), although not all puncta conform to thispattern (presumably due to the presence of homotypic fila-ments, unipolar filaments [22], filaments that are partially outof the TIRF zone, bipolar filaments with a unipolar distributionof one isoform, and/or heterotypic filaments where the signalfor one isoform is below the level of detection; see Experi-mental Procedures for further discussion). Heterotypic

Non-muscle myosin II (NMII) is a conserved force-producing cytoskeletal enzyme with important but poorly understood roles in cell migration. To investigate myosin heavy chain (MHC) phosphorylation roles in 3D migration, we expressed GFP-tagged NMIIA wild-type or mutant constructs in cells depleted of endogenous NMIIA protein. We find that individual mutation or double mutation of Ser-1916 or Ser-1943 to alanine potently blocks recruitment of GFP-NM-IIA filaments to leading edge protrusions in 2D, and this in turn blocks maturation of anterior focal adhesions. When placed in 3D collagen gels, cells expressing wild-type GFP MHC-IIA behave like parental cells, displaying robust and active formation and retraction of protrusions. However, cells depleted of NMIIA or cells expressing the mutant GFP MHC-IIA display severe defects in invasion and in stabilizing protrusions in 3D. These studies reveal an NMIIA-specific role in 3D invasion that requires competence for NMIIA phosphorylation at Ser-1916 and Ser-1943. In sum, these results demonstrate a critical and previously unrecognized role for NMIIA phosphorylation in 3D invasion.

Wound healing in the skin is an important and complex process that involves 3-dimensional tissue reorganization, including matrix and chemokine-triggered cell migration, paracrine signaling, and matrix remodeling. The molecular signals and underlying mechanisms that stimulate myosin II activity during skin wound healing have not been elucidated. To begin understanding the signaling pathways involved in the activation of myosin II in this process, we have evaluated myosin II activation in migrating primary human keratinocytes in response to scratch wounding in vitro. We report here that myosin II activation and recruitment to the cytoskeleton in wounded keratinocytes are biphasic. Post-wounding, a rapid phosphorylation of myosin II regulatory light chain (RLC) occurs with resultant translocation of myosin IIA to the cell cortex, far in advance of the later polarization and cell migration. During this acute-phase of myosin II activation, pharmacological approaches reveal p38-MAP kinase and cytosolic calcium as having critical roles in the phosphorylation driving cytoskeletal assembly. Although p38-MAPK has known roles in keratinocyte migration, and known roles in leading-edge focal complex dynamics, to our knowledge this is the first report of p38-MAPK acting as an upstream activator of myosin II phosphorylation and assembly during any type of wound response.

During cell division, the mechanisms by which myosin II is recruited to the contractile ring are not fully understood. Much recent work has focused on a model in which spatially restricted de novo filament assembly occurs at the cell equator via localized myosin II regulatory light chain (RLC) phosphorylation, stimulated by the RhoA-activating centralspindlin complex. Here, we show that a recombinant myosin IIA protein that assembles constitutively and is incapable of binding RLC still displays strong localization to the furrow in mammalian cells. Furthermore, this RLC-deficient myosin II efficiently drives cytokinesis, demonstrating that centralspindlin-based RLC phosphorylation is not necessary for myosin II localization during furrowing. Myosin II truncation analysis further reveals two distinct myosin II tail properties that contribute to furrow localization: a central tail domain mediating cortical furrow binding to heterologous binding partners and a carboxyl-terminal region mediating co-assembly with existing furrow myosin IIA or IIB filaments.

Non-muscle myosin 2 (NM2) is a major force-producing, actin-based motor in mammalian non-muscle cells, where it plays important roles in a broad range of fundamental biological processes, including cytokinesis, cell migration, and epithelial barrier function.. This breadth of function at the tissue and cellular levels suggests extensive diversity and differential regulation of NM2 bipolar filaments, the major, if not sole, functional form of NM2s in vivo. Previous in vitro, cellular and animal studies indicate that some of this diversity is supported by the existence of multiple NM2 isoforms. Moreover, two recent studies have shown that these isoforms can co-assemble to form heterotypic filaments, further expanding functional diversity. In addition to isoform co-assembly, cells may differentially regulate NM2 function via isoform-specific expression, RLC phosphorylation, MHC phosphorylation or regulation via binding partners. Here, we provide a brief summary of NM2 filament assembly, summarize the recent findings regarding NM2 isoform co-assembly, consider the mechanisms cells might utilize to differentially regulate NM2 isoforms, and review the data available to support these mechanisms.

Tumor necrosis factor α (TNF-α) elicits its biological activities through activation of TNF receptor 1 (TNFR1, also known as p55) and TNFR2 (also known as p75). The activities of both receptors are required for the TNF-α–induced proinflammatory response. The adaptor protein TNFR-associated factor 2 (TRAF2) is critical for either p55- or p75-mediated activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, as well as for target gene expression. Here, we identified nonmuscle myosin II (myosin) as a binding partner of p75. TNF-α–dependent signaling by p75 and induction of target gene expression persisted for substantially longer in cells deficient in myosin regulatory light chain (MRLC, a component of myosin) than in cells replete in myosin. In resting endothelial cells, myosin was bound constitutively to the intracellular region of p75, a region that overlaps with the TRAF2-binding domain, and TNF-α caused the rapid dissociation of myosin from p75. At early time points after exposure to TNF-α, p75 activated Rho-associated kinase 1 (ROCK1). Inhibition of ROCK1 activity blocked TNF-α–dependent phosphorylation of MRLC and the dissociation of myosin from p75. ROCK1-dependent release of myosin was necessary for the TNF-α–dependent recruitment of TRAF2 to p75 and for p75-specific activation of NF-κB and MAPK signaling. Thus, our findings have revealed a previously uncharacterized, noncanonical regulatory function of myosin in cytokine signaling.

Background Phosphorylation of non-muscle myosin II regulatory light chain (RLC) at Thr18/Ser19 is well established as a key regulatory event that controls myosin II assembly and activation, both in vitro and in living cells. RLC can also be phosphorylated at Ser1/Ser2/Thr9 by protein kinase C (PKC). Biophysical studies show that phosphorylation at these sites leads to an increase in the Km of myosin light chain kinase (MLCK) for RLC, thereby indirectly inhibiting myosin II activity. Despite unequivocal evidence that PKC phosphorylation at Ser1/Ser2/Thr9 can regulate myosin II function in vitro, there is little evidence that this mechanism regulates myosin II function in live cells. Results The purpose of these studies was to investigate the role of Ser1/Ser2/Thr9 phosphorylation in live cells. To do this we utilized phospho-specific antibodies and created GFP-tagged RLC reporters with phosphomimetic aspartic acid substitutions or unphosphorylatable alanine substitutions at the putative inhibitory sites or the previously characterized activation sites. Cell lines stably expressing the RLC-GFP constructs were assayed for myosin recruitment during cell division, the ability to complete cell division, and myosin assembly levels under resting or spreading conditions. Our data shows that manipulation of the activation sites (Thr18/Ser19) significantly alters myosin II function in a number of these assays while manipulation of the putative inhibitory sites (Ser1/Ser2/Thr9) does not. Conclusions These studies suggest that inhibitory phosphorylation of RLC is not a substantial regulatory mechanism, although we cannot rule out its role in other cellular processes or perhaps other types of cells or tissues in vivo.

SummaryClass-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.