Myosin IIA drives neurite retraction

Neuritic extension is the resultant of two vectorial processes: outgrowth and retraction. Whereas myosin IIB is required for neurite outgrowth, retraction is driven by a motor whose identity has remained unknown until now. Preformed neurites in mouse Neuro-2A neuroblastoma cells undergo immediate retraction when exposed to isoform-specific antisense oligonucleotides that suppress myosin IIB expression, ruling out myosin IIB as the retraction motor. When cells were preincubated with antisense oligonucleotides targeting myosin IIA, simultaneous or subsequent addition of myosin IIB antisense oligonucleotides did not elicit neurite retraction, both outgrowth and retraction being curtailed. Even during simultaneous application of antisense oligonucleotides against both myosin isoforms, lamellipodial spreading continued despite the complete inhibition of neurite extension, indicating an uncoupling of lamellipodial dynamics from movement of the neurite. Significantly, lysophosphatidate- or thrombin-induced neurite retraction was blocked not only by the Rho-kinase inhibitor Y27632 but also by antisense oligonucleotides targeting myosin IIA. Control oligonucleotides or antisense oligonucleotides targeting myosin IIB had no effect. In contrast, Y27632 did not inhibit outgrowth, a myosin IIB-dependent process. We conclude that the conventional myosin motor, myosin IIA, drives neurite retraction.     

Myosin IIC: a third molecular motor driving neuronal dynamics

Neuronal dynamics result from the integration of forces developed by molecular motors, especially conventional myosins. Myosin IIC is a recently discovered nonsarcomeric conventional myosin motor, the function of which is poorly understood, particularly in relation to the separate but coupled activities of its close homologues, myosins IIA and IIB, which participate in neuronal adhesion, outgrowth and retraction. To determine myosin IIC function, we have applied a comparative functional knockdown approach by using isoform-specific antisense oligodeoxyribonucleotides to deplete expression within neuronally derived cells. Myosin IIC was found to be critical for driving neuronal process outgrowth, a function that it shares with myosin IIB. Additionally, myosin IIC modulates neuronal cell adhesion, a function that it shares with myosin IIA but not myosin IIB. Consistent with this role, myosin IIC knockdown caused a concomitant decrease in paxillin-phospho-Tyr118 immunofluorescence, similar to knockdown of myosin IIA but not myosin IIB. Myosin IIC depletion also created a distinctive phenotype with increased cell body diameter, increased vacuolization, and impaired responsiveness to triggered neurite collapse by lysophosphatidic acid. This novel combination of properties suggests that myosin IIC must participate in distinctive cellular roles and reinforces our view that closely related motor isoforms drive diverse functions within neuronal cells.     

Segregation and activation of myosin IIB creates a rear in migrating cells

We have found that MLC-dependent activation of myosin IIB in migrating cells is required to form an extended rear, which coincides with increased directional migration. Activated myosin IIB localizes prominently at the cell rear and produces large, stable actin filament bundles and adhesions, which locally inhibit protrusion and define the morphology of the tail. Myosin IIA forms de novo filaments away from the myosin IIB–enriched center and back to form regions that support protrusion. The positioning and dynamics of myosin IIA and IIB depend on the self-assembly regions in their coiled-coil C terminus. COS7 and B16 melanoma cells lack myosin IIA and IIB, respectively; and show isoform-specific front-back polarity in migrating cells. These studies demonstrate the role of MLC activation and myosin isoforms in creating a cell rear, the segregation of isoforms during filament assembly and their differential effects on adhesion and protrusion, and a key role for the noncontractile region of the isoforms in determining their localization and function.     

Myosin II activity regulates neurite outgrowth and guidance in response to chondroitin sulfate proteoglycans

Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix in the CNS that inhibit axonal regeneration after CNS injury. Signaling pathways in neurons triggered by CSPGs are still largely unknown. In this study, using well-characterized in vitro assays for neurite outgrowth and neurite guidance, we demonstrate a major role for myosin II in the response of neurons to CSPGs. We found that the phosphorylation of myosin II regulatory light chains is increased by CSPGs. Specific inhibition of myosin II activity with blebbistatin allows growing neurites to cross onto CSPG-rich areas and increases the length of neurites of neurons growing on CSPGs. Using specific gene knockdown, we demonstrate selective roles for myosin IIA and IIB in these processes. Time lapse microscopy and immunocytochemistry demonstrated that CSPGs also inhibit cell adhesion and cell spreading. Inhibition of myosin II selectively accelerated neurite initiation without altering cell adhesion and spreading on CSPGs.     

Neurite Differentiation Is Modulated in Neuroblastoma Cells Engineered for Altered Acetylcholinesterase Expression

Abstract: Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level ( p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.     

Growth cones contain myosin II bipolar filament arrays

Nonmuscle myosin II is among the most abundant forms of myosin in nerve growth cones. At least two isoforms of myosin II (A and B) that have overlapping but distinct distributions are found in growth cones. It appears that both myosin IIA and IIB may be necessary for normal nerve outgrowth and motility, but the molecular interactions responsible for their activity remain unclear. For instance, it is unknown if these myosin II isoforms produce bipolar "minifilaments" in growth cones similar to those observed in other nonmuscle cells. To determine if minifilaments are present in growth cones, we modified the electron microscopy preparative procedures used to detect minifilaments in other cell types. We found structures that appeared very similar to bipolar minifilaments found in noneuronal cells. They also labeled with antibodies to either myosin IIA or IIB. Thus, the activity of myosin II in growth cones is likely to be similar to that in other nonmuscle cells. Bipolar filaments interacting with oppositely oriented actin filaments will produce localized contractions or exert tension on actin networks. This activity will be responsible for the myosin II dependent motility in growth cones.     

Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta.

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.     

Nonmuscle myosin IIA and IIB have distinct functions in the exocytosis-dependent process of cell membrane repair

Vesicle generation, recruitment, and exocytosis are essential for repairing disruptions of cell membranes. The functions of nonmuscle myosin IIA and IIB in this exocytotic process of membrane repair were studied by the antisense technique. Knockdown of myosin IIB suppressed wound-induced exocytosis and the membrane resealing process. Knockdown of myosin IIA did not suppress exocytosis at an initial wound and had no inhibitory effect on the resealing at initial wounds but did inhibit the facilitated rate of resealing normally found at repeated wounds made at the same site. COS-7 cells, which lack myosin IIA, did not show the facilitated response of membrane resealing to a repeated wound. S91 melanoma cells, a mutant cell line lacking myosin Va, showed normal membrane resealing and normal facilitated responses. We concluded that myosin IIB was required for exocytosis and therefore cell membrane repair itself and that myosin IIA was required in facilitation of cell membrane repair at repeated wounds. Myosin IIB was primarily at the subplasmalemma cortex and myosin IIA was concentrated at the trans-Golgi network consistent with their distinct roles in vesicle trafficking in cell membrane repair.     

Of Mice and Men

Non-muscle myosin II has diverse functions in cell contractility, morphology, cytokinesis and migration. Mammalian cells have three isoforms of non-muscle myosin II, termed IIA, IIB and IIC, encoded by three different genes. These isoforms share considerable homology and some overlapping functions, yet they exhibit differences in enzymatic properties, subcellular localization, molecular interaction and tissue distribution 1-6. Our studies have focused on the IIA isoform, and they reveal unique regulatory roles in cell-cell adhesion and cell migration that are associated with cross-talk of the actomyosin system with microtubules. In humans, various mutations in the MYH9 gene that encodes the myosin IIA heavy chain cause autosomal dominant disease, whereas in mice, the complete deficiency is embryonic lethal but heterozygous mice are nearly normal. We discuss here the differences between mouse and human phenotypes and how the wealth of mechanistic knowledge about myosin II based on in vitro studies and mouse models can help us understand the molecular and cellular pathophysiology of myosin IIA deficiency in humans.     

Asymmetric distribution of myosin IIB in migrating endothelial cells is regulated by a rho-dependent kinase and contributes to tail retraction

All vertebrates contain two nonmuscle myosin II heavy chains, A and B, which differ in tissue expression and subcellular distributions. To understand how these distinct distributions are controlled and what role they play in cell migration, myosin IIA and IIB were examined during wound healing by bovine aortic endothelial cells. Immunofluorescence showed that myosin IIA skewed toward the front of migrating cells, coincident with actin assembly at the leading edge, whereas myosin IIB accumulated in the rear 15-30 min later. Inhibition of myosin light-chain kinase, protein kinases A, C, and G, tyrosine kinase, MAP kinase, and PIP3 kinase did not affect this asymmetric redistribution of myosin isoforms. However, posterior accumulation of myosin IIB, but not anterior distribution of myosin IIA, was inhibited by dominant-negative rhoA and by the rho-kinase inhibitor, Y-27632, which also inhibited myosin light-chain phosphorylation. This inhibition was overcome by transfecting cells with constitutively active myosin light-chain kinase. These observations indicate that asymmetry of myosin IIB, but not IIA, is regulated by light-chain phosphorylation mediated by rho-dependent kinase. Blocking this pathway inhibited tail constriction and retraction, but did not affect protrusion, suggesting that myosin IIB functions in pulling the rear of the cell forward.     

Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.     

Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts.

We investigated the localization of non-muscle myosin II isoforms and mono- (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non-motile MRC-5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non-motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration.     

Specific features of neuronal size and shape are regulated by tropomyosin isoforms

Spatially distinct populations of microfilaments, characterized by different tropomyosin (Tm) isoforms, are present within a neuron. To investigate the impact of altered tropomyosin isoform expression on neuronal morphogenesis, embryonic cortical neurons from transgenic mice expressing the isoforms Tm3 and Tm5NM1, under the control of the beta-actin promoter, were cultured in vitro. Exogenously expressed Tm isoforms sorted to different subcellular compartments with Tm5NM1 enriched in filopodia and growth cones, whereas the Tm3 was more broadly localized. The Tm5NM1 neurons displayed significantly enlarged growth cones accompanied by an increase in the number of dendrites and axonal branching. In contrast, Tm3 neurons displayed inhibition of neurite outgrowth. Recruitment of Tm5a and myosin IIB was observed in the peripheral region of a significant number of Tm5NM1 growth cones. We propose that enrichment of myosin IIB increases filament stability, leading to the enlarged growth cones. Our observations support a role for different tropomyosin isoforms in regulating interactions with myosin and thereby regulating morphology in specific intracellular compartments.     

Neuronal cell adhesion, mediated by the heparin-binding neuroregulatory factor midkine, is specifically inhibited by chondroitin sulfate E. Structural ans functional implications of the over-sulfated chondroitin sulfate

The heparin-binding neurotrophic factor midkine (MK) has been proposed to mediate neuronal cell adhesion and neurite outgrowth promotion by interacting with cell-surface heparan sulfate. We have observed that over-sulfated chondroitin sulfate (CS) D and CS-E show neurite outgrowth-promoting activity in embryonic day (E) 18 rat hippocampal neurons (Nadanaka, S., Clement, A., Masayama, K., Faissner, A., and Sugahara, K. (1998) J. Biol. Chem. 273, 3296-3307). In the present study, various CS isoforms were examined for their ability to inhibit the MK-mediated cell adhesion of cortical neuronal cells in comparison with heparin from porcine intestine and heparan sulfate from bovine kidney. E17-18 rat cortical neuronal cells were cultured on plates coated with recombinant MK in a grid pattern. The cells attached to and extended their neurites along the MK substratum. Cell adhesion was inhibited by squid cartilage over-sulfated CS-E as well as by heparin, but not by heparan sulfate or other CS isoforms. Direct interactions of MK with various glycosaminoglycans were then evaluated using surface plasmon resonance, showing that CS-E bound MK as strongly as heparin, followed by other over-sulfated CS isoforms, CS-H and CS-K. Furthermore, E18 rat brain extracts showed an E disaccharide unit, GlcUAbeta1-3GalNAc(4,6-O-disulfate). These findings indicate that CS chains containing the E unit as well as heparin-like glycosaminoglycans may be involved in the expression and/or modulation of the multiple neuroregulatory functions of MK such as neuronal adhesion and migration and promotion of neurite outgrowth.     

Expression of NCAM containing VASE in neurons can account for a developmental loss in their neurite outgrowth response to NCAM in a cellular substratum

Binding of the neural cell adhesion molecule (NCAM) in neurons to NCAM on non-neuronal cells can stimulate axonal growth. A developmentally regulated loss of this response is associated with the insertion of 10 amino acids (called VASE) into the fourth Ig domain in up to 50% of the NCAM receptors in neurons. In the present study we have transfected PC12 cells with the major neuronal isoforms of human NCAM and tested cells expressing these isoforms for their ability to respond to NCAM in a cellular substratum. Whereas both the 140- and 180-kD isoforms of NCAM can act as functional receptors for neurite outgrowth, the presence of the VASE sequence in a minority of the receptors specifically inhibited this response. A synthetic peptide containing the VASE sequence inhibits neurite outgrowth from PC12 cells and primary neurons stimulated by NCAM. The same peptide has no effect on integrin dependent neurite outgrowth or neurite outgrowth stimulated by N-cadherin or L1. We discuss the possibility that the VASE peptide inhibits the NCAM response by preventing NCAM from binding to the FGF receptor in the plasma membrane.     

Diphosphorylation of regulatory light chain of myosin IIA is responsible for proper cell spreading

The actomyosin cytoskeleton plays prominent roles in cell spreading and migration. To address the roles of myosin II isoforms and to estimate the region where the myosin IIs are activated in spreading cells, we examined the immunolocalization of myosin II isoforms and phosphorylated RLCs in the spreading MRC-5 cells. We observed the formation of actin ring-like structure at the base of the lamella. Both myosin IIA and IIB were predominantly localized there. Myosin IIA and diphosphorylated RLC were distributed outside of the region where myosin IIB and monophosphoryated RLC were distributed predominantly. Inhibition of Rho-kinase resulted in the disappearance of the diphosphorylation of RLC, moreover, it accelerated the rate of cell spreading and induced an aberrant cell shape at later stage of spreading. These results indicate that diphosphorylation of RLCs of myosin IIA by Rho-kinase in lamella is responsible for the cell to spread properly.     

Cell adhesion molecules NgCAM and axonin-1 form heterodimers in the neuronal membrane and cooperate in neurite outgrowth promotion

The axonal surface glycoproteins neuronglia cell adhesion molecule (NgCAM) and axonin-1 promote cell-cell adhesion, neurite outgrowth and fasciculation, and are involved in growth cone guidance. A direct binding between NgCAM and axonin-1 has been demonstrated using isolated molecules conjugated to the surface of fluorescent microspheres. By expressing NgCAM and axonin-1 in myeloma cells and performing cell aggregation assays, we found that NgCAM and axonin-1 cannot bind when present on the surface of different cells. In contrast, the cocapping of axonin-1 upon antibody-induced capping of NgCAM on the surface of CV- 1 cells coexpressing NgCAM and axonin-1 and the selective chemical cross-linking of the two molecules in low density cultures of dorsal root ganglia neurons indicated a specific and direct binding of axonin- 1 and Ng-CAM in the plane of the same membrane. Suppression of the axonin-1 translation by antisense oligonucleotides prevented neurite outgrowth in dissociated dorsal root ganglia neurons cultured on an NgCAM substratum, indicating that neurite outgrowth on NgCAM substratum requires axonin-1. Based on these and previous results, which implicated NgCAM as the neuronal receptor involved in neurite outgrowth on NgCAM substratum, we concluded that neurite outgrowth on an NgCAM substratum depends on two essential interactions of growth cone NgCAM: a trans-interaction with substratum NgCAM and a cis-interaction with axonin-1 residing in the same growth cone membrane.     

Dystrophin Dp71 is required for neurite outgrowth in PC12 cells

To determine the role of Dp71 in neuronal cells, we generated PC12 cell lines in which Dp71 protein levels were controlled by stable transfection with either antisense or sense constructs. Cells expressing the antisense Dp71 RNA (antisense-Dp71 cells) contained reduced amounts of the two endogenous Dp71 isoforms. Antisense-Dp71 cells exhibited a marked suppression of neurite outgrowth upon the induction with NGF or dibutyryl cyclic AMP. Early responses to NGF-induced neuronal differentiation, such as the cessation of cell division and the activation of ERK1/2 proteins, were normal in the antisense-Dp71 cells. On contrary, the induction of MAP2, a late differentiation marker, was disturbed in these cells. Additionally, the deficiency of Dp71 correlated with an altered expression of the dystrophin-associated protein complex (DAPC) members alpha and beta dystrobrevins. Our results indicate that normal expression of Dp71 is essential for neurite outgrowth in PC12 cells and constitute the first direct evidence implicating Dp71 in a neuronal function.     

Myosin II Motor Proteins with Different Functions Determine the Fate of Lamellipodia Extension during Cell Spreading

Non-muscle cells express multiple myosin-II motor proteins myosin IIA, myosin IIB and myosin IIC transcribed from different loci in the human genome. Due to a significant homology in their sequences, these ubiquitously expressed myosin II motor proteins are believed to have overlapping cellular functions, but the mechanistic details are not elucidated. The present study uncovered a mechanism that coordinates the distinctly localized myosin IIA and myosin IIB with unexpected opposite mechanical roles in maneuvering lamellipodia extension, a critical step in the initiation of cell invasion, spreading, and migration. Myosin IIB motor protein by localizing at the front drives lamellipodia extension during cell spreading. On the other hand, myosin IIA localizes next to myosin IIB and attenuates or retracts lamellipodia extension. Myosin IIA and IIB increase cell adhesion by regulating focal contacts formation in the spreading margins and central part of the spreading cell, respectively. Spreading cells expressing both myosin IIA and myosin IIB motor proteins display an organized actin network consisting of retrograde filaments, arcs and central filaments attached to focal contacts. This organized actin network especially arcs and focal contacts formation in the spreading margins were lost in myosin IIÂ cells. Surprisingly, myosin II$\widehat{B}$ cells displayed long parallel actin filaments connected to focal contacts in the spreading margins. Thus, with different roles in the regulation of the actin network and focal contacts formation, both myosin IIA and IIB determine the fate of lamellipodia extension during cell spreading.     

Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.