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At least 19 recordsLinked to original sources

PubMed2002

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.

Actin Cytoskeleton↗

PubMed2006

Protein kinase Cgamma regulates myosin IIB phosphorylation, cellular localization, and filament assembly.

Nonmuscle myosin II is an important component of the cytoskeleton, playing a major role in cell motility and chemotaxis. We have previously demonstrated that, on stimulation with epidermal growth factor (EGF), nonmuscle myosin heavy chain II-B (NMHC-IIB) undergoes a transient phosphorylation correlating with its cellular localization. We also showed that members of the PKC family are involved in this phosphorylation. Here we demonstrate that of the two conventional PKC isoforms expressed by prostate cancer cells, PKCbetaII and PKCgamma, PKCgamma directly phosphorylates NMHC-IIB. Overexpression of wild-type and kinase dead dominant negative PKCgamma result in both altered NMHC-IIB phosphorylation and subcellular localization. We have also mapped the phosphorylation sites of PKCgamma on NMHC-IIB. Conversion of the PKCgamma phosphorylation sites to alanine residues, reduces the EGF-dependent NMHC-IIB phosphorylation. Aspartate substitution of these sites reduces NMHC-IIB localization into cytoskeleton. These results indicate that PKCgamma regulates NMHC-IIB phosphorylation and cellular localization in response to EGF stimulation.

Actomyosin↗

PubMed2001

Myosin IIB is required for growth cone motility.

Growth cones are required for the forward advancement and navigation of growing axons. Modulation of growth cone shape and reorientation of the neurite are responsible for the change of outgrowth direction that underlies navigation. Change of shape involves the reordering of the cytoskeleton. Reorientation of the neurite requires the generation of tension, which is supplied by the ability of the growth cone to crawl on a substrate. The specific molecular mechanisms responsible for these activities are unknown but are thought to involve actomyosin-generated force combined with linkage to the cell surface receptors that are responsible for adhesion (Heidemann and Buxbaum, 1998). To test whether myosin IIB is responsible for the force generation, we quantified shape dynamics and filopodial-mediated traction force in growth cones from myosin IIB knock-out (KO) mice and compared them with neurons from normal littermates. Growth cones from the KO mice spread less, showed alterations in shape dynamics and actin organization, and had reduced filopodial-mediated traction force. Although peak traction forces produced by filopodia of KO cones were decreased significantly, KO filopodia occasionally developed forces equivalent to those in the wild type. This indicates that other myosins participate in filopodial-dependent traction force. Therefore, myosin IIB is necessary for normal growth cone spreading and the modulation of shape and traction force but acts in combination with other myosins for some or all of these activities. These activities are essential for growth cone forward advancement, which is necessary for outgrowth. Thus outgrowth is slowed, but not eliminated, in neurons from the myosin IIB KO mice.

Acrylic Resins↗

PubMed2000

B2 exon splicing of nonmuscle myosin heavy chain IIB is differently regulated in developing and adult rat brain.

Two isoforms of nonmuscle myosin heavy chain IIB (MHC-IIB) are generated by alternative splicing; MHC-IIB(B2) differs from MHC-IIB(DeltaB2) by the insertion of B2 exon cassette near the actin binding region. Here we examined expressions of the two splice variants in developing and adult rat brains by in situ hybridization with isoform-specific oligonucleotide probes. In adult, MHC-IIB(DeltaB2) mRNA was highly expressed in neurons of the cerebral cortex, hippocampus, and cerebellum, whereas MHC-IIB(B2) mRNA was mainly distributed in the brainstem and cerebellum, with the highest level in Purkinje cells. During development, MHC-IIB(DeltaB2) mRNA was predominantly expressed in various regions of embryonic and neonatal brains, whereas MHC-IIB(B2) mRNA was low during embryonic stages. Up-regulation of MHC-IIB(B2) started in the cerebellum during early postnatal stages when dendritogenesis and synaptogenesis occur actively in Purkinje cells. We further employed immunofluorescence using two antibodies (one recognizing both splicing variants and another specific to MHC-IIB(B2)), and found similar and dense localization in cell bodies and dendrites of Purkinje cells. Therefore, splicing of the B2 exon cassette undergoes distinct temporal and spatial regulations in the brain in vivo, and the different exon usage seems unlikely to affect the somato-dendritic localization of MHC-IIB.

Alternative Splicing↗

PubMed2003

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.

Amides↗

PubMed2006

Targeted disruption of N-RAP gene function by RNA interference: a role for N-RAP in myofibril organization.

N-RAP is a muscle-specific protein concentrated in myofibril precursors during sarcomere assembly and at intercalated disks in adult heart. We used RNA interference to achieve a targeted decrease in N-RAP transcript and protein levels in primary cultures of embryonic mouse cardiomyocytes. N-RAP transcript levels were decreased by approximately 70% within 2 days following transfection with N-RAP specific siRNA. N-RAP protein levels steadily decreased over several days, reaching approximately 50% of control levels within 6 days. N-RAP protein knockdown was associated with decreased myofibril assembly, as assessed by alpha-actinin organization into mature striations. Transcripts encoding N-RAP binding proteins associated with assembling or mature myofibrils, such as alpha-actinin, Krp1, and muscle LIM protein, were expressed at normal levels during N-RAP protein knockdown, and alpha-actinin and Krp-1 protein levels were also unchanged. Transcripts encoding muscle myosin heavy chain and nonmuscle myosin heavy chain IIB were also expressed at relatively normal levels. However, decreased N-RAP protein levels were associated with dramatic changes in the encoded myosin proteins, with muscle myosin heavy chain levels increasing and nonmuscle myosin heavy chain IIB decreasing. N-RAP transcript and protein levels recovered to normal by days 6 and 7, respectively, and the changes in myofibril organization and myosin heavy chain isoform levels were reversed. Our data indicate that we can achieve transient N-RAP protein knockdown using the RNA interference technique and that alpha-actinin organization into myofibrils in cardiomyocytes is closely linked to N-RAP protein levels. Finally, N-RAP protein levels regulate the balance between nonmuscle myosin IIB and muscle myosin by post-trancriptional mechanisms.

Actinin↗

PubMed2007

Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB.

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.

Actins↗

PubMed2003

Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb.

Methods for cell type specific targeted intracellular delivery of proteins in vivo remain limited. A murine monoclonal anti-dsDNA antibody, mAb 3E10, was selectively transported into skeletal muscle cells in vivo. The antibody bound a 200 kDa protein only found in lysates of skeletal muscle by Western blotting. The 200 kDa protein was purified from muscle lysate by antibody affinity chromatography and identified as the skeletal muscle specific heavy chain of myosin IIb by electrospray mass spectrometry. Antibody binding specificity for myosin IIb was demonstrated in Western blots by binding myosin in skeletal muscle lysates from mice null for myosin IId but not in mice null for myosin IIb. Myosin IIb is implicated in the specific targeting of mAb 3E10 to skeletal muscle.

Amino Acid Sequence↗

PubMed2003

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.

Actins↗

PubMed2006

Myosin light chain kinase colocalizes with nonmuscle myosin IIB in myofibril precursors and sarcomeric Z-lines of cardiomyocytes.

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility involving actin and myosin II. MLCK is widely present in vertebrate tissues including the myocardium. However, the role of MLCK in cardiomyocyte function is not known. Previous attempts to gain insight into possible roles and identify potential molecular partners were disappointing and equivocal due to cross reactivity of early antibodies with striated muscle MLCK, which has a different genetic locus and a divergent amino acid sequence from the above mentioned enzyme. Using an immunofluorescence approach and a panel of antibodies directed against MLCK, cytoskeletal, and sarcomeric proteins, we localized MLCK to myofibril precursors and Z-lines of sarcomeres in embryonic and adult cardiomyocytes. The same structures contained nonmuscle myosin IIB implicating this protein as a possible target of MLCK. Our results suggest a role for MLCK in cardiomyocyte differentiation and contraction through regulation of nonmuscle myosin IIB.

Animals↗

PubMed2004

BDNF rescues myosin heavy chain IIB muscle fibers after neonatal nerve injury.

Neonatal sciatic nerve injury is known to result in an extensive loss of lumbar motor neurons as well as the disappearance of their respective muscle fibers in the hindlimb musculature. The loss of motor neurons and muscle fibers can be prevented by immediate administration of target-derived neurotrophic factors to the site of injury. In the present study, we investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. We have shown that combined administration of CNTF and BDNF prevented the loss of motor units after neonatal nerve injury and contributed to the maintenance of muscle mass. Importantly, this combined neurotrophin regimen also prevented the disappearance of muscle fibers that express myosin heavy chain IIB (MyHC IIB) in both EDL and TA muscles 3 mo after neonatal sciatic nerve crush. In parallel studies, we observed a higher level of BDNF in EDL muscle during the critical period of development when motor neurons are highly susceptible to target removal. Given our previous findings that combined administration of CNTF with neurotrophin-3 (NT-3) or neurotrophin-4/5 (NT-4/5) did not result in the rescue of MyHC IIB fibers in EDL, the present results show the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons and of MyHC IIB muscle fibers during neonatal development of the neuromuscular system.

Animals↗

PubMed2006

Distinct roles of nonmuscle myosin II isoforms in the regulation of MDA-MB-231 breast cancer cell spreading and migration.

Initial stages of tumor cell metastasis involve an epithelial-mesenchyme transition that involves activation of amoeboid migration and loss of cell-cell adhesion. The actomyosin cytoskeleton has fundamental but poorly understood roles in these events. Myosin II, an abundant force-producing protein, has roles in cell body translocation and retraction of the posterior of the cell during migration. Recent studies have suggested that this protein may also have roles in leading edge protrusive events. The metastasis-promoting protein metastasin-1, a regulator of myosin II assembly, colocalizes with myosin IIA at the leading edge of cancer cells, suggesting direct roles for myosin II in metastatic behavior. We have assessed the roles of specific myosin II isoforms during lamellar spreading of MDA-MB-231 breast cancer cells on extracellular matrix. We find that the two major myosin II isoforms IIA and IIB are both expressed in these cells, and both are recruited dramatically to the lamellar margin during active spreading on fibronectin. There is also a transient increase in regulatory light chain phosphorylation that correlates the recruitment of myosin IIA and myosin IIB into this spreading margin. Pharmacologic inhibition of myosin II or myosin light chain kinase dramatically reduced spreading. Depletion of myosin IIA via small interfering RNA impaired migration but enhanced lamellar spreading, whereas depletion of myosin IIB impaired not only migration but also impaired initial rates of lamellar spreading. These results indicate that both isoforms are critical for the mechanics of cell migration, with myosin IIB seeming to have a preferential role in the mechanics of lamellar protrusion.

Breast Neoplasms↗

PubMed2003

Retrograde flow rate is increased in growth cones from myosin IIB knockout mice.

Growth cones of myosin-IIB-knockout mice have reduced outgrowth rates and traction force. There is a close relationship between traction force, retrograde flow and forward advance of growth cones. All three activities appear to be at least partially myosin dependent. Therefore, we have now tested for differences in retrograde flow rates between growth cones from myosin-IIB-knockout mice and their normal littermates. By placing nerve-growth-factor-coated silica beads on the surface of growth cones with laser tweezers, or by tracking GFP-myosin IIA spots, we found that the retrograde flow rate was increased more than two fold in the knockout growth cones compared with the wild type. These data suggest that both myosin IIA and IIB normally contribute to retrograde flow and the properties of the flow are strongly influenced by myosin IIB because of its location and abundance. However, in the absence of myosin IIB, myosin IIA takes over this function. The change in retrograde flow rate may reflect the difference in functional properties of these two myosins. Knockout growth cones also exhibited reduced stability of lamellipodia, possibly as a partial consequence of this increased retrograde flow rate. In addition, microtubules penetrated a shorter distance into filopodia, which suggests that the increase in flow rate may adversely affect the microtubule-dependent maturation of filopodia. Taken together these data support the idea that the forward advance of the growth cone is myosin II dependent and involves multiple myosin II isoforms.

Animals↗

PubMed2003

F-actin and myosin II binding domains in supervillin.

Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We find that the supervillin N terminus binds directly to myosin II, as well as to F-actin. Three F-actin-binding sites were mapped to sequences within amino acids approximately 280-342, approximately 344-422, and approximately 700-830. Sequences with combinations of these sites promote F-actin cross-linking and/or bundling. Supervillin amino acids 1-174 specifically interact with the S2 domain in chicken gizzard myosin and nonmuscle myosin IIA (MYH-9) but exhibit little binding to skeletal muscle myosin II. Direct or indirect binding to filamin also was observed. Overexpression of supervillin amino acids 1-174 in COS7 cells disrupted the localization of myosin IIB without obviously affecting actin filaments. Taken together, these results suggest that supervillin may mediate actin and myosin II filament organization at cholesterol-rich membrane domains.

Actins↗

PubMed1999

Developmentally regulated expression of a nonmuscle myosin heavy chain IIB inserted isoform in rat brain.

The alternatively spliced isoform of the nonmuscle myosin II heavy chain B (MHC-B) with an insert of 21 amino acids at the 50- to 20-kDa junction of the globular region of myosin has been demonstrated to be expressed specifically in the central nervous system (CNS) in chicken. To explore the role of this B2 inserted isoform (MHC-B(B2)), immunoblot and immunoprecipitation analyses were performed using specific antibodies and extracts from rat tissues. MHC-B(B2) is present throughout the CNS, but is less abundant in the cerebrum and not expressed in the olfactory lobe at all. In the developing rat brain, MHC-B(B2) is expressed from postnatal day 10 (P10) in the cerebellum and increases markedly from P14. The appearance of MHC-B(B2) in the cerebrum (P28) is later than in the cerebellum. Additionally, we show that myosin IIB(B2) is homodimeric in its heavy chain subunit composition. These results suggest that myosin IIB(B2) might participate in cell motility in the neuronal cells of the mature CNS.

Alternative Splicing↗

PubMed2003

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.

3T3 Cells↗

PubMed2003

Kinetic mechanism of non-muscle myosin IIB: functional adaptations for tension generation and maintenance.

Besides driving contraction of various types of muscle tissue, conventional (class II) myosins serve essential cellular functions and are ubiquitously expressed in eukaryotic cells. Three different isoforms in the human myosin complement have been identified as non-muscle class II myosins. Here we report the kinetic characterization of a human non-muscle myosin IIB subfragment-1 construct produced in the baculovirus expression system. Transient kinetic data show that most steps of the actomyosin ATPase cycle are slowed down compared with other class II myosins. The ADP affinity of subfragment-1 is unusually high even in the presence of actin filaments, and the rate of ADP release is close to the steady-state ATPase rate. Thus, non-muscle myosin IIB subfragment-1 spends a significantly higher proportion of its kinetic cycle strongly attached to actin than do the muscle myosins. This feature is even more pronounced at slightly elevated ADP levels, and it may be important in carrying out the cellular functions of this isoform working in small filamentous assemblies.

Actins↗

PubMed2001

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.

Cell Movement↗