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Nat Rev Mol Cell Biol. Author manuscript; available in PMC Mar 8, 2010.
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Non-muscle myosin II takes centre stage in cell adhesion and migration

Abstract

Non-muscle myosin II (NM II) is an actin-binding protein that has actin cross-linking and contractile properties and is regulated by the phosphorylation of its light and heavy chains. The three mammalian NM II isoforms have both overlapping and unique properties. Owing to its position downstream of convergent signalling pathways, NM II is central in the control of cell adhesion, cell migration and tissue architecture. Recent insight into the role of NM II in these processes has been gained from loss-of-function and mutant approaches, methods that quantitatively measure actin and adhesion dynamics and the discovery of NM II mutations that cause monogenic diseases.

Myosins constitute a superfamily of motor proteins that play important parts in several cellular processes that require force and translocation13. Recent analyses of genomic databases have yielded an increasing number of myosin classes in eukaryotic cells4,5. Myosin molecules can walk along, propel the sliding of or produce tension on actin filaments. This requires energy, which is provided by the hydrolysis of ATP, and requires myosins to have catalytic sites with ATPase activity. Myosin catalytic sites are usually found in the amino-terminal (head) region of the molecule, and they are often activated when myosin binds to actin. The carboxy-terminal region of some myosins binds to and moves cargo in a cell, whereas the C-terminal domains of other myosins self-associate into filaments, which allows their heads to tether actin filaments and exert tension. Myosins can also act indirectly through actin to bring adhesion-related proteins, such as integrins, or signal transduction molecules into close proximity2,3.

Most myosins belong to class II and, together with actin, make up the major contractile proteins of cardiac, skeletal and smooth muscle, in which the sliding crossbridges that connect thick myosin filaments with thin actin filaments provide the force to, for example, pump blood, lift objects and expel babies6,7. Importantly, myosin II molecules that resemble their muscle counterparts, with respect to both structure and function, are also present in all non-muscle eukaryotic cells811. Like muscle myosin II, non-muscle myosin II (NM II) molecules are comprised of three pairs of peptides: two heavy chains of 230 kDa, two 20 kDa regulatory light chains (RLCs) that regulate NM II activity and two 17 kDa essential light chains (ELCs) that stabilize the heavy chain structure (FIG. 1a). Although these myosins are referred to as ‘non-muscle’ myosin IIs to distinguish them from their muscle counterparts, they are also present in muscle cells, where they have distinct functions during skeletal muscle development and differentiation12, as well as in the maintenance of tension in smooth muscle13,14.

Figure 1
Domain structure of NM II

NM II has a fundamental role in processes that require cellular reshaping and movement, such as cell adhesion, cell migration and cell division. NM II can use its actin cross-linking and contractile functions, which are regulated by phosphorylation and the ability of NM II to form filaments, to regulate the actin cytoskeleton.

In this Review, we provide an overview of the structure and regulation of NM II, with emphasis on its central role in cell adhesion and cell migration, and also outline the relationship between NM II mutations and disease.

Domain structure of NM II

The two globular head domains of NM II contain a binding site for both ATP and actin and they are followed by neck regions, each of which binds the two functionally different light chains. The neck domain acts as a lever arm to amplify head rotation while the chemical energy of ATP is converted into the mechanical movement of the myosin head1. This neck domain is followed by a long α-helical coiled coil, which forms an extended rod-shaped domain that effects dimerization between the heavy chains and terminates in a relatively short non-helical tail (FIG. 1a). The rod domains of NM II self-associate to form bipolar filaments (anti-parallel arrays of myosin molecules), which are considerably smaller than those found in cardiac and skeletal muscle15,16 (FIG. 1b).

Three different genes in mammalian cells (myosin heavy chain 9 (MYH9), MYH10 and MYH14) encode the NM II heavy chain (NMHC II) proteins (NMHC IIA, NMHC IIb and NMHC IIC, respectively), although there is only one NMHC II gene, zipper, in Drosophila melanogaster17. We refer to the whole myosin II molecule (heavy chains and light chains) as NM II and the heavy chains alone as NMHCs. The NMHC isoform determines the NM II isoform, which are named NM IIA, NM IIB or NM IIC, accordingly. For NM II motors to retain their normal, native and active conformations, the light chains must be bound to the heavy chains. Deletion of a specific NMHC II results in the loss of that NM II isoform.

The mammalian heavy chain pre-mRNAs that are transcribed in humans and mice from MYH10 and MYH14 undergo alternative splicing, predominantly in neuronal tissue, which increases the total number of expressed NMHC II proteins to nine18. Although there is evidence for alternative splicing of the MYH9 transcripts at locations homologous to those in MYH10 and MYH14, it is not known whether these transcripts are translated19. The light chains are encoded by a different set of genes, which can also undergo alternative splicing, and there is currently no known specificity of light chains for particular NMHC IIs. The two light chain pairs are very tightly, but non-covalently, bound to α-helical stretches of each heavy chain. Despite having a high degree of homology, particularly in their actin-binding globular heads, the myosin isoforms are spatially segregated in some areas of cells, but clearly overlap in others20,21. As detailed below, some cellular functions are isoform-specific, whereas others are redundant22,23.

The short, non-helical tails of NMHC II proteins differ sufficiently among the three mammalian isoforms that peptides can be synthesized to mimic these sequences for the generation of isoform-specific antibodies24. There is no evidence for heterodimer formation between the three NMHC II isoforms, and when immunoprecipitation experiments are performed on a mixture of isoforms with specific antibodies only the antibody-specific isoform is detected24. Two important kinetic properties that differ among the NM II isoforms are the actin-activated Mg2+-ATPase activity (the increase in ATP hydrolysis by myosin when bound to actin) and the duty ratio (the time that myosin is bound to actin in a force-generating state). NM IIA has the highest rate of ATP hydrolysis of the three NM II isoforms and it propels actin filaments more rapidly than NM IIB and NM IIC25. NM IIb has a significantly higher duty ratio than NM IIA26,27. NM IIB also has an extremely high affinity for ADP when compared to other myosins, and the release of ADP from NM IIB is markedly slowed by backward strain on the myosin head (the force exerted on myosin heads by the actin filaments, opposite to the direction of myosin head movement)28. Thus, NM IIB is particularly well suited to exert tension on actin filaments for longer periods of time and with less expenditure of energy than NM IIA. Indeed, NM IIB has recently been shown to play an ancillary role in smooth muscle contraction, in which it may be responsible for a portion of the tension maintenance during tonic contractions13. These differences in kinetic properties between NM IIA and NM IIB might help to explain why expressing NMHC IIA under the control of the endogenous MYH10 promoter can rescue some, but not all, of the abnormalities found in NM IIB-ablated mice29.

Whereas the different enzymatic and motor activities of NM IIs reside in their N-terminal domain, the C-terminal rod and non-helical tail determine the assembly of myosin filaments and the intracellular localization of the NM II isoforms. Two assembly-competent domains in the NM IIb rod that have a crucial role in filament formation30, and two regions in the NM IIb rod that are involved in isoform-specific filament assembly, have been identified31.

Experiments using chimaeras of NM IIA and NM IIB have shown that the intracellular localization of these isoforms is determined by the C-terminal 179–190 amino acids, which contain one of the assembly-competent domains and the non-helical tail32,33.

Regulation of NM II activity

In contrast to skeletal and cardiac myosins, which are regulated by a separate set of proteins that are bound to the actin filaments, the regulation of Mg2+-ATP hydrolysis and filament formation of NM II involves the reversible phosphorylation of specific amino acids present in the pair of 20 kDa RLCs and the heavy chains. The function of the ELC pair is to stabilize the NMHC and there is no evidence that they undergo reversible phosphorylation.

Regulation of NM II by RLC phosphorylation

The regulation of NM IIA, NM IIB, NM IIC and smooth muscle myosin, but not cardiac or mammalian skeletal muscle myosin, depends on the reversible phosphorylation of the RLC on ser19. This phosphorylation event greatly increases the Mg2+-ATPase activity of myosin in the presence of actin34 by controlling the conformation of the myosin heads35. However, phosphorylation has little or no effect on the affinity of myosin for actin36. Thr18 can also be phosphorylated, and diphosphorylation of the RLC on ser19 and Thr18 often occurs in cultured cells37. The additional phosphorylation of Thr18 in the presence of phosphorylated ser19 (REF. 38) further increases the total actin-activated Mg2+-ATPase activity at sub-saturating actin concentrations, but does not affect the myosin vmax (the maximal actin-activ ated Mg2+-ATPase activity of myosin at saturating actin concentrations) or the rate of movement of actin filaments in vitro39.

There is compelling evidence from in vitro experiments that phosphorylation of the RLC also regulates the assembly of NM II filaments40. In the presence of ATP, unphosphorylated NM II folds into a compact conformation, in which one head blocks the second head of the same molecule through an asymmetrical interaction (FIG. 1a; assembly-incompetent form)35,4144. The tail also folds at two points and interacts with the heads to further compact the molecule and prevent filament assembly. Phosphorylation of RLCs disrupts the head–head and head–tail interactions and causes the compact, faster sedimenting (10s) molecule to form an elongated slower sedimenting (6s) species (FIG. 1a), which forms bipolar filaments at physiological ionic strengths (FIG. 1b). Although these dramatic changes can be demonstrated in solution, there is no firm evidence that they occur in vivo. The formation of assemblies comprising 14–20 myosin molecules in bipolar filaments promotes the interaction of myosin filaments with actin filaments (FIG. 1b).

More than a dozen kinases have been reported to phosphorylate the RLCs of NM II (FIG. 2), including myosin light chain kinase (MLCK; also known as MYLK), Rho-associated, coiled coil-containing kinase (ROCK), citron kinase, leucine zipper interacting kinase (ZIPK; also known as DAPK3) and myotonic dystrophy kinase-related CDC42-binding kinase (MRCK; also known as CDC42bP)6,34,45,46. These kinases phosphorylate RLCs on ser19, Thr18 or both, to relieve the inhibition imposed on the myosin molecule by unphosphorylated RLCs and the head–head interaction outlined above. However, the activating signals for these kinases differ. Ca2+–calmodulin activates MLCK, which seems to be specific for the NM II RLCs, whereas the small GTP-binding protein RHOA activates ROCK and citron kinase. unlike MLCK, ROCK and citron kinase phosphorylate several substrates in addition to the RLCs of NM II. For example, although ROCK can phosphorylate RLCs directly, it primarily acts to inhibit the major myosin phosphatase, protein phosphatase 1 (PP1), which is responsible for the dephosphorylation of NM II47. Phosphorylation of the regulatory subunit myosin phosphatase-targeting sub unit 1 (MYPT1; also known as PPP1R12A) of this trimeric phosphatase inactivates the enzymatic activity, resulting in increased NM II phosphorylation and activation. MLCK, at least in some cells, is more peripherally localized, wherase ROCK is more central48,49. This differential localization means that the actomyosin structures in the cell centre (such as stress fibres and mature focal adhesions) are much more stable than those at the periphery, which are more responsive to upstream stimuli. Protein kinase C (PKC) differs from the above kinases in that it phosphorylates RLCs on ser1, ser2 and Thr9, which renders the RLC a poorer substrate for MLCK and therefore decreases the activity of NM II50 (FIG. 2). For example, PKC phosphorylation of the RLC on ser1 or ser2 initiated by stimulation of platelet-derived growth factor receptor inhibits NM II activity to promote the reorganization of actomyosin filaments51. As noted below, in addition to phosphorylating the RLCs, PKC also phosphorylates the heavy chains.

Figure 2
Regulation of NM II activation and filament formation

Regulation of NM II by NMHC phosphorylation

Although phosphorylation of NMHC II plays an important part in regulating the activity of Dictyostelium discoideum myosin II (reviewed in REF. 52), the roles in regulating the structure and activity of mammalian NM II are still being defined. There are several phosphorylation sites near the C-termini of NMHCs, in both the coiled-coil domain and the non-helical tail, including sites that are phosphorylated by PKC53, casein kinase II (CK II)54 and transient receptor potential melastatin 7 (TRPM7)55 (FIG. 2; see below). In each case phosphorylation of the NMHC either dissociates myosin filaments or prevents their formation in vitro. A number of laboratories have reported that ser1917 in rat NMHC IIA (equivalent to ser1916 in human NMHC IIA) is phosphorylated by PKC, and phosphorylation of this site by PKCβ correlates with exocytosis in mast cells56,57. ser1943 in the non-helical NMHC tail is a substrate for phosphorylation by CK II, and phosphorylation of this residue, or of ser1917 by PKC, inhibits the assembly of NM IIA rods into filaments54.

Phosphorylation of NM IIA also affects the binding of s100A4 (also known as MTs1) to the NMHC. This protein, which is a member of the s100 family of Ca2+-binding proteins, promotes the invasion of metastatic tumour cells. binding of s100A4 to NMHC IIA prevents filament formation, but s100A4–NMHC IIA binding is blocked by the phosphorylation of ser1943 by CK II58. An alpha kinase from the same family as D. discoideum NMHC kinase A, TRPM7, and its close homologue TRPM6, phosphorylate NM IIB and NM IIC at several sites in the non-helical tails59. Although the effects of these phosphorylation events on filament assembly have not yet been studied, the more specific phosphorylations catalysed by TRPM7 in the helical portion of the rod in NM IIA at Thr1800, ser1803 and ser1808 seem to decrease filament formation and alter the subcellular localization of NM IIA55. As yet, there is no evidence for the endogenous phosphorylation of these sites.

Phosphorylation of NM IIB by aPKCζ, which is regulated by p21-activated kinase 1 (PAK1), was studied in a prostate cancer cell line. This phosphorylation, which occurs on ser1937 in the non-helical tail region of NMHC IIB, is stimulated by epidermal growth factor (EGF) and leads to slower filament assembly in cultured cells53. Recently, phosphorylation of both the non-helical tail and the rod of the NMHC has been reported for NM IIC. Phosphorylation increases the solubility of NM IIC and also has an effect on cellular localization60. Thus, it is clear that considerable regulation of NM II occurs through phosphorylation of the heavy chains, particularly in the tail region (FIG. 2). The continued characterization of the roles of these different phosphorylation events on NM II function and isoform-specific regulation should produce important new insights.

NM II in cell migration, adhesion and polarity

NM II is an important regulator of adhesion and polarity in cell migration. These processes involve the dynamic remodelling of the actin cytoskeleton and the interaction of the cell with its environment. Each of the NM II isoforms affects these processes differently, as discussed below.

NM II regulates protrusion and cell migration

In migrating cells, actin organizes into several distinct structures and its polymerization in cellular protrusions drives cell migration. Protrusions generally contain two actin-based structures: the lamellipodium and the lamellum61. Different classes of regulatory molecules organize actin in these two structures. The actin nucleator Arp2/3 generates the lamellipodium. In vitro, Arp2/3 binds to the sides of actin filaments and generates branches at a fixed angle (70°) to form a dendritic network62. However, the in vivo data are still under debate. A recent study has reported actin filaments with variable angles (15°–90°) in the lamellipodium, which challenges the dendritic network notion63. The relative positioning of the lamellipodium and the lamellum is also unclear. It was recently proposed that these two actin networks overlap at the leading edge, with the lamellipodium superimposed onto the lamellum64, but a more traditional view is that the lamellipodium is spatially anterior to the lamellum61. In the lamellum, actin filaments coalesce into thicker bundles (FIG. 3a). The lamellipodium and the lamellum are kinetically different: the lamelli podium is distinguished by a fast retrograde flow of actin, whereas the lamellum exhibits slower retrograde flow. The convergent zone between the two is characterized by active depolymerization of the dendritic network and the reorganization of actin64,65 (FIG. 3b).

Figure 3
Multiple roles of NM II in cell migration

NM II does not reside in or seem to play a part in the physical organization of the lamellipodium, but it can affect the net rate of cellular protrusion22,64,66 (FIG. 3b). When NM II is knocked out, knocked down by small interfering RNA or inhibited with blebbistatin, large actin bundles are not observed in the lamellum, whereas the lamellipodium remains intact64. In many cells, advancement of the protrusion is interrupted by NM II-generated pauses67 that are absent when myosin is inhibited or deleted22.

one hypothesis for the role of NM II in protrusion formation is that NM II generates the retrograde flow of actin in the lamellum, which is connected to the lamellipodium64,68 (FIG. 3b). Inhibition of NM II activity with blebbistatin, or genetic deletion of NM II, greatly decreases the rate of actin retrograde flow in the lamellum64,66 and inhibits coalescence of actin into proto-bundles at the lamellipodium–lamellum interface69,70, which increases protrusiveness22,66,71 (FIG. 3b). NM II-generated retrograde flow counters the actin polymerization-mediated advancement of the leading edge and thereby reduces the observed protrusion rate. Thus, periodic contractions of the lamellipodium could cyclically inhibit the protrusion rate. In this model, the protrusion rate is the difference between the actin polymerization and the retrograde flow rates. Adhesion modulates this balance by coupling actin to the substratum, thereby creating traction points that counter actin retrograde flow72. such a mechanism presumably increases the rate of protrusion by diverting the forces created by the retrograde flow of the actin to the substratum. Thus, the linkage between actin and the substratum constitutes a ‘molecular clutch’. These observations, first made in neuronal growth cones but later extended to many other cell types, constitute the clutch hypothesis7274, which integrates adhesion, retrograde flow and actin polymerization.

NM II in integrin-mediated adhesion

NM II is dispensable for the assembly and disassembly of nascent adhesions inside the lamellipodium75,76. However, it is required for their subsequent maturation, which involves elongation and growth. Adhesions mature along thin actin bundles that originate near the transition zone at the lamellipodium and lamellum interface75 (FIG. 3b). The probability that a nascent adhesion in the lamellipodium matures, rather than disassembles, seems to depend on the level of active NM II75. However, the mechanism by which adhesions elongate and grow, and how NM II mediates this maturation, is not completely understood. The actin bundling function of NM II seems to be sufficient, at least for the initial stages of adhesion maturation, because motor-deficient NM II mutants that still bind to actin support growth and elongation of adhesions75.

An integrin–actin linkage translates the effect of NM II to adhesions and mediates adhesion formation and maturation. Different adhesion proteins seem to participate in this linkage, including α-actinin, vinculin and talin (FIG. 4). There are two non-exclusive hypotheses for how NM II mediates adhesion maturation. one is that NM II bundles actin filaments, and as a consequence adhesion proteins at the ends of these actin filaments are brought together and clustered. This increases molecular inter actions between adhesion proteins and results in increased integrin avidity (the combined strength of multiple integrin–ligand interactions) and signalling. For example, the actomyosin-driven binding of vinculin to talin induces integrin clustering77. The second hypothesis is that NM II-generated force induces conformational changes that expose cryptic binding or activation sites in key adhesion components7880. Talin, an actin–integrin linkage protein, binds vinculin when mechanically stretched79 and is required to translate NM II-generated forces to the substratum81 (FIG. 4). The adhesion scaffold protein CRK-associated substrate (p130CAS; also known as BCAR1) is also activated when stretched78. Furthermore, both ends of the adhesive linkage respond to force. Fibronectin undergoes conformational changes induced by mechanical force that result in the exposure of its cryptic sites82, and integrins are activated by tension80. Finally, NM II itself can also undergo force-dependent conformational changes83. Interestingly, the force-dependent modulation of mechanoreactive NM II molecules also impinges on other signalling molecules that are not mechanosensitive themselves. It has recently been proposed that NM II also controls EGF-induced paxillin phosphorylation and its subsequent dephosphorylation, which regulates cellular protrusion and retraction84. In addition, zyxin associates and dissociates from adhesions in response to extracellular forces85, and focal adhesion kinase (FAK), a prominent adhesion-associated tyrosine kinase, is required for durotaxis (that is, the tendency of cells to migrate towards stiffer substrates)86.

Figure 4
NM II in integrin-mediated adhesion

NM II is also an integral part of the cellular response to mechanical stimulation. It reacts to mechanical stimuli through cellular signalling pathways that regulate its activation. In this way, NM II provides a convergence point for external and cell-generated forces. For example, application of external forces produces post-translational modifications such as phosphorylation, or conformational changes in different signalling molecules, which inhibit protrusion formation and lead to adhesion maturation and actin filament bundling87. Conversely, cells in which NM II is inhibited do not respond to external forces88. In addition, cells sense substrate pliability, in part through the activation of NM II by RLC phosphorylation89,90. When cells are plated on low-pliability substrates, NM II activation is low, actin is not highly bundled and adhesions are small. As rigidity increases, NM II activation increases, actin is visibly bundled and adhesions become large and elongated.

Adhesive signalling in NM II activation

NM II influences adhesive signalling through clustering and/or conformational changes, but adhesive signalling also controls NM II activation (FIG. 4). For example, integrin activation induces Tyr phosphorylation of the adhesion adaptor paxillin on Tyr31 and Tyr118 and of FAK on Tyr397. These phosphorylations trigger the activation and recruitment to adhesions of signalling intermediates such as the p130CAS–CRK and G protein-coupled receptor kinase interacting ArfGAP (GIT)–β-Pix (also known as ARHGEF7) complexes, relaying activation signals to Rho GTPases, particularly Rac (reviewed in REFS 9193). Among other functions, Rac triggers actin polymerization through the Arp2/3 complex and also inhibits NM II activation (FIG. 4). other signalling pathways activated by adhesion have the opposite effect and promote NM II activation through RHOA94,95. Rac is activated by signals generated in small adhesions close to the leading edge that actively undergo turnover and reassemble. RHOA activation mediates actin filament formation and adhesion maturation as the protrusion moves away and the adhesions become more central. This induces further activation of NM II and the coalescence of thick actomyosin bundles. During adhesion maturation, Rac signalling decreases and RHOA-mediated activation of NM II increases, which results in enhanced actomyosin bundling87,89,96.

NM II is a dual regulator of protrusion, through its effects on actin retrograde flow and adhesion-generated signalling22,66. Increased activation of NM II results in large actin bundles and stable adhesions, decreased signalling to Rac and decreased protrusion. Lower levels of active NM II result in less actin bundling and increased protrusion. This can explain, in part, the underlying differences in migration among different cell types. Specifically, highly migratory cells such as leukocytes do not exhibit large adhesions97, probably reflecting low levels of NM II activation or an intrinsic inability to re arrange their actin cytoskeleton into large bundles, whereas slow moving cells such as fibroblasts have adhesions that tend to mature into large, elongated structures as a result of NM II activation and robust actin bundling.

NM II activation promotes adhesion maturation through its actin bundling and contractile activities. Cycles of Rac and Rho activation inactivate and activate NM II, resulting in protrusion and actin bundling, respectively. The role of NM II in adhesion depends on its ability to exert force on adhesions, even though it is not physically present in the adhesions but attaches to the actin bundles with which adhesions are associated22. Thus, NM II influences adhesion from a distance.

NM II determines the polarity of migrating cell

Directional migration requires an initial symmetry break and the polarization of the migratory machinery. There is strong evidence that NM II contractility initiates symmetry breaking by forming the prospective rear of the cell98100, which allows for protrusion to occur at the opposite end. The signals leading to specification of the cell rear and protrusion are spatially segregated101.

The leading protrusion resides at the cell front, and the cell rear often adopts a defined morphology, such as flat and parallel to the leading edge in keratocytes, or lagging and triangular in some fibroblasts. The molecular composition and organization of these front and rear compartments is distinct102, particularly with regards to the actin cytoskeleton and the adhesions in these regions. The tail region is usually comprised of larger actin filament bundles that support adhesion disassembly and retraction of the rear, and large, stable adhesions that do not signal to Rac. By contrast, adhesions at the front are smaller and more dynamic and mediate robust Rac signalling, which drives protrusion.

NM II activation is essential for the formation of a defined rear compartment. NM II localizes to the posterior part of migrating leukocytes and D. discoideum103,104. Here, it creates stable adhesions and highly bundled actin, which prevent protrusion and thereby generate non-protrusive areas that constitute the sides and the trailing edge of the migrating cell33,98.

In migrating fibroblasts, the NM II isoforms have different roles in cell polarization. NM IIA is dynamic and assembles actomyosin bundles in protrusions22,66. By contrast, NM IIB incorporates into preformed actin bundles and remains stationary, defining the centre and rear of the migrating cell33. In this manner, the cooperative functions of NM IIA and NM IIB induce big, non-dynamic actomyosin structures that define the non-protrusive parts of the cell (the centre and the trailing edge), whereas dynamic filaments in protruding regions of the cell are comprised of NM IIA alone33. NM IIA specifically mediates retraction of the trailing edge during migration105 (FIG. 3c). When NM IIA is inhibited, cells become elongated owing to their failure to retract105,106.

In addition to differences in actin and adhesion organization and function, the position of the nucleus, the microtubule-organizing centre (MTOC) and the Golgi apparatus are also hallmarks of migratory cell polarization. The MTOC and Golgi apparatus reposition in front of the nucleus towards the direction of protrusion107. Data from experiments in which NM II activity was inhibited by blebbistatin reveal that NM II also regulates nucleus repositioning108 (FIG. 3d). NM IIB seems to play a prominent part in nuclear dynamics during migration as cells depleted of NM IIB exhibit multidirectional protrusions109, lack a clear front and back, have a mislocalized Golgi and MTOC, exhibit inappropriate rotation of the nucleus and fail to reposition the nucleus properly during polarized migration22. One hypothesis is that NM IIB directly anchors the perinuclear actin at the nucleus by interacting with nuclear transmembrane receptors such as nesprins, which are involved in nuclear redistribution during cell migration110,111. Another possibility is that NM IIB provides tension or structural rigidity to the perinuclear actin and this force prevents inappropriate rotation of the nucleus and moves it forwards (FIG. 3d).

NM II in cell–cell adhesion and morphogenesis

NM II is not only pivotal in controlling integrin-mediated adhesion and migration, it also regulates epithelial cell adhesion, polarization and morphogenesis. Well-defined cell–cell junctions are a key feature of epithelial sheets and represent a different type of adhesive structure that is controlled by NM II. Although these cell–cell junctions use cadherins as the main adhesion receptors, they contain scaffolds and signalling intermediates analogous to those found in integrin-mediated complexes. In aggregates, epithelial cells have apical and basolateral regions as well as integrin-based adhesions to the extracellular matrix (ECM) at the basal surface (FIG. 5). Epithelial cell sheets can move as multicellular cohorts, with the leading cells showing protrusions and the trailing cells retracting, or they can detach from the sheet and move away as single cells. In free edges of the sheet, the migrating cells undergo a polarity switch from apicobasal (FIG. 5) to front–back (FIG. 3).

Figure 5
Roles of NM II in epithelial cell polarization

NM II controls the formation and stability of the cell–cell junctions. NM IIA is required for cadherin clustering112,113 (FIG. 5) and the stability of the cell–cell junction. The initiation of the contact is driven by actin polymerization114, and NM II-driven contractility mediates the later stages of contact formation (compaction). Active RHOA and phosphorylated RLCs localize to the periphery of the junction and drive the contraction and compaction of the cell–cell contact114 (FIG. 5). RHOA activation, which activates NM II, also mediates contact inhibition of movement (that is, the inhibition of protrusion by cell–cell contact)115. NM IIA probably plays a similar part during the formation of T cell–antigen-presenting cell conjugates in antigenic presentation116.

NM II-driven mechanisms also govern the three-dimensional organization of epithelial tissues. In three dimensions, the force applied to a single cell is potentially multi-directional, rather than planar or one-dimensional. The rigidity of the cellular microenvironment is crucial. There is much evidence that NM II is part of the mechanism by which the cell both generates and responds to force. Three dimensional responses have been studied in Xenopus laevis and D. melanogaster during early embryonic development and organogenesis. In both systems, the same general principles of regulation of NM II apply. In D. melanogaster, NM II (NMHC encoded by zipper) is activated by phosphorylation of the RLCs (encoded by spaghetti squash), which is under the control of Rho kinase (ROK)117119. NM II is also implicated in epithelial processes involving contraction, such as gastrulation120, trachea formation121,122, ventral furrow formation123,124, dorsal closure117 and morphogenesis of the vertebrate neural tube124126, as well as in the re organization of cell contacts in the plane of the epithelium during convergent extension127,128. In D. melanogaster eye imaginal disc formation, morphogenetic signalling is under the control of Hedgehog, which activates NM II through the RHOA–ROK pathway129.

Finally, NM II provides the tension and traction that cells use to form and remodel a functional ECM during tissue morphogenesis and wound repair. This includes NM II-powered collagen fibre remodelling130 and fibronectin fibrillogenesis82,131. Recently, a link between cell–cell contact organization and fibrillogenesis was proposed. Actomyosin reorganization driven by cadherins translates into ECM remodelling through integrins, suggesting that cadherin-generated signals regulate integrin activation by modulating cytoskeletal tension132 (FIG. 5).

NM II mutations and disease

An early indication that NM IIA, NM IIB and NM IIC have different roles during development came from studies in mice showing that disruption of the genes encoding the heavy chains of these proteins (MYH9, MYH10 and MYH14, respectively) leads to different phenotypes. NMHC IIA germline-ablated mice fail to form a polarized visceral endoderm and show defects in cell–cell adhesion that result in death by embryonic day (E) 6.5, before organ formation133. Mice in which NMHC IIB has been deleted or mutated survive to E14.5 and show cardiac and brain defects, which reflects the enrichment of the NM IIB isoform in these organs in wild-type mice134,135. As noted below, the brain defects were due to failures in both cell adhesion and cell migration.

To decipher the role of NM II in adhesion and migration, several investigators used mutants that mimic the mutations found in humans in MYH9 and MYH14 and expressed the mutant NMHC IIA and NMHC IIC proteins in mice, D. melanogaster and cultured cells. To date, almost 40 mutations in NMHC IIA, distributed among the head and rod domains, have been reported to cause MYH9-related diseases136 (TABLE 1). All of the patients with these diseases were heterozygous for MYH9 mutations. one of the most common abnormalities in patients with MYH9-related diseases is associated with dysfunction of the blood platelets, which play an important part in blood clotting and clot retraction, and affected patients suffer from macrothrombocytopenia. Platelets from patients with mutated NM IIA have an abnormal cytoskeletal composition and fail to form and/or reorganize cytoskeletal structures on stimulation with the thrombin receptor activating peptide137. The initial shape change and internal contraction of the platelets following stimulation requires NM IIA activity, which in turn is activated by RLC phosphorylation138,139. Baculoviral-expressed catalytic fragments (heavy meromyosins) of NM IIA mutants that mimic the human mutations in the motor domain (N93K and R702C in NMHC IIA), have a reduced actin-activated Mg2+-ATPase activity and an inability to translocate actin filaments in vitro140. It is therefore likely that impairment of NM IIA motor function contributes to the platelet defects in these patients.

Table 1
The 39 MYH9 mutations associated with MYH9-related diseases*

Both haploinsufficiency and the ability of mutant myosin to interfere with wild-type myosin (dominant-negative effects) have been proposed as mechanisms by which mutated NM IIA leads to MYH9-related diseases. A reduction in wild-type NM IIA, accompanied by no or very low levels of the mutant form of NM IIA, has been observed in platelets and their precursors (megakaryocytes) from patients with MYH9-related diseases. However, both haploinsufficiency and dominant-negative effects have been reported in different tissues from the same patient. Haploinsufficiency was shown to be the mechanism by which mutated NM IIA leads to MYH9-related diseases in platelets, and dominant-negative effects were shown to be the mechanism in granulocytes141,142. Mice with NM IIA specifically ablated from megakaryocytes have thrombocytopenia with large platelets similar to those in patients with MYH9-related diseases, as well as defects in cytoskeletal reorganization143,144.

So far there have been no reports of mutations in MYH10, which encodes NMHC IIB, associated with human disease. To establish a putative mouse model for such diseases, mice carrying the R709C mutation in NMHC IIB (a homologue of the R702C mutation in human NMHC IIA) have been generated145. Homozygous mutant mice die by E14.5 and show major morphogenetic defects, including a failure to close the ventral body wall, resulting in a cleft palate, ectopia cordis and a large omphalocele, as well as defects in heart and diaphragm development (X.M. and R.S.A., unpublished observations). These abnormalities are reminiscent of a rare human syndrome known as Pentalogy of Cantrell146.

Mice ablated for NM IIB, in contrast to mice with point mutations in NMHC IIB, develop hydrocephalus (in which circulation of the cerebral spinal fluid is blocked), one of the most common congenital diseases that affects humans (1–3 per 1,000 live births). This is because loss of NM IIB causes disruption of apical cell–cell adhesion of the neuroepithelial cells that border the spinal canal147. Interestingly, this phenotype can be rescued by expression of the NMHC IIB point mutant R709C, which has a motor that is deficient in enzyme activity, or by using gene substitution to replace NM IIB with NM IIA29,147. NM IIA is normally not detected in the neuroepithelial apical adhesion complex and demonstrates markedly increased enzymatic motor properties compared with NM IIB, as well as a decreased duty ratio (see above). Therefore it is likely that the structural properties of NM IIB, rather than its enzymatic motor activity, are essential for maintaining the integrity of cell–cell adhesion in the neuroepithelial cells that line the spinal canal10.

Finally, five mutations in MYH14, which encodes NMHC IIC, have been reported to occur in both the myosin head and tail domains. All of these mutations result in deafness148,149. The in vivo function of NM IIC is largely unknown and mice ablated for NM IIC show no obvious abnormalities (X.M. and R.S.A., unpublished observations).

The pathophysiological roles of NM II are still not fully understood. Although there is no evidence for heterodimer formation among the NMHC isoforms, there is evidence for colocalization of NM II isoforms in filaments. This raises the interesting possibility that a mutant isoform might interfere with a second (or third) wild-type isoform.

Studies from genetically manipulated mouse models demonstrate that NM II is involved in cell–cell adhesion and cell migration in vivo. In maintaining the integrity of cell–cell adhesions, NM IIA and NM IIB are functionally interchangeable and this function seems to be independent of their actin-activated Mg2+-ATPase motor activity. In driving cell migration, however, the ATPase activity of the myosin motor is essential and NM IIA and NM IIB cannot substitute for each other.

Concluding remarks

Through its effects on actin bundling and contractility, NM II acts as a master integrator of the processes that drive cell migration and adhesion. It is also an important end point on which many signalling pathways converge, largely through Rho GTPases. NM II itself is tightly regulated at different levels, including at the level of folding, myosin filament assembly and disassembly, actin binding and ATPase and motor activity. The regulation of the actin cytoskeleton by NM II controls multiple interrelated processes, such as migration, cell–cell and cell–matrix adhesion, cell differentiation, tissue morphogenesis and development. Furthermore, NM II is at one end of the regulatory feedback loops that control the activation of NM II’s own upstream signalling pathways, including the conformational activation of adhesion molecules and actin organization. The importance of NM II is highlighted by the profound and varied physiological effects of genetic deletion of NM II isoforms in animal models and the high number of naturally occurring mutations in the genes encoding the NMHC II proteins that cause human disease.

The recent interest in the cellular and tissue functions of NM II has produced many revealing and insightful observations, although many basic and translational questions remain. For example, the spatiotemporal regulation of NM II by subcellular localization and activation of its regulating kinases in different cells and tissues has important ramifications in controlling the NM II function but needs further investigation. Emerging evidence strongly indicates that mechanical forces probably remodel the tumour cell microenvironment through NM II to affect tumour progression and metastasis150, but the precise mechanism by which NM II responds to and generates the microenvironment remains to be elucidated. In addition, the notion that NM II is a central integrator of external force during cell and tissue differentiation, as well as migration, opens up the possibility of manipulating both the rigidity of the microenvironment and the expression and activation of NM II to guide cell proliferation and differentiation during tissue regeneration and stem cell transplantation. In this way, the manipulation of NM II and its regulators constitutes a potentially valuable approach in regenerative medicine.

FURTHER INFORMATION

Alan Rick Horwitz’s homepage: http://people.virginia.edu/~afh2n/

Robert S. Adelstein’s homepage: http://dir.nhlbi.nih.gov/labs/lmc

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

Acknowledgements

The authors apologize for the numerous studies left out owing to journal limits on the number of references. We thank M. A. Conti and J. R. Sellers (National Heart, Lung and Blood Institute (NHLBI)) for helpful suggestions and critical reading of the manuscript. This work was supported by the US National Institutes of Health (NIH) grant GM23244, the Cell Migration Consortium grant U54-GM064346 (A.R.H.), and the Division of Intramural Research, NHLBI, NIH (R.S.A.).

Glossary

Actin filament
A strand of polymerized globular actin subunits that winds around another strand to form a helix. Actin filaments are one of the three major cytoskeletal elements of a cell, along with microtubules and intermediate filaments.
Integrin
One of a large family of heterodimeric transmembrane proteins that functions as a receptor for ECM or cell adhesion molecules.
Coiled coil
A structural domain that can mediate oligomerization. The myosin coiled-coil rod domain contains two α-helices that twist around each other to form a supercoil.
Tonic contractions
Sustained muscular contractions that develop slowly and show a prolonged phase of relaxation.
Actomyosin filaments
Produced when bipolar myosin filaments interact with polymerized actin filaments to exert tension or produce movement.
Lamellipodium
A 1–2 µm-wide band that is made up of a network of dendritic actin filaments and forms the outer edge of a cell protrusion.
Lamellum
The cell region immediately behind the lamellipodium, characterized by the absence of dendritic actin and the presence of longer, bundled actin filaments and slow retrograde flow.
Blebbistatin
A small-molecule inhibitor with high affinity for myosin II that blocks myosin in an actin-detached state.
Pliability
The mechanical properties of the cellular environment. The environment can show low pliability (that is, be elastic or compliant) or high pliability (that is, be rigid or stiff).
Microtubule-organizing centre
A eukaryotic cell structure from which microtubules emanate. During mitosis, the MTOC organizes the mitotic spindle.
Cadherin
A member of a family of type I transmembrane receptors that mediate cell–cell adhesion through homophilic interactions.
Convergent extension
A phase of gastrulation in which layers of cells intercalate (converge) and become longer (extend). Extension is driven by a rearrangement of the cells of the ventral part of the epithelium, which converge towards the ventral midline.
Fibrillogenesis
A cell-induced reorganization of the surrounding ECM molecules into bundled fibres.
Macrothrombocytopenia
A condition that is characterized by enlarged blood platelets that are approximately the size of red blood cells, are reduced in number and result in prolonged bleeding times.
Haploinsufficiency
A state in which the loss of only one allele of a gene detectably disables the encoded protein’s function.
Ectopia cordis
A congenital displacement of the heart outside the thoracic cavity and chest wall.
Omphalocele
A protrusion that occurs at birth, whereby part of the intestine protrudes through a large defect in the abdominal wall at the umbilicus.

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