Replacement of nonmuscle myosin II-B with II-A rescues brain but not cardiac defects in mice

The purpose of these studies was to learn whether one isoform of nonmuscle myosin II, specifically nonmuscle myosin II-A, could functionally replace a second one, nonmuscle myosin II-B, in mice. To accomplish this, we used homologous recombination to ablate nonmuscle myosin heavy chain (NMHC) II-B by inserting cDNA encoding green fluorescent protein (GFP)-NMHC II-A into the first coding exon of the Myh10 gene, thereby placing GFP-NMHC II-A under control of the endogenous II-B promoter. Similar to B(-)/B(-) mice, most B(a*)/B(a*) mice died late in embryonic development with structural cardiac defects and impaired cytokinesis of the cardiac myocytes. However, unlike B(-)/B(-) mice, 15 B(a*)/B(a*) mice of 172 F2 generation mice survived embryonic lethality but developed a dilated cardiomyopathy as adults. Surprisingly none of the B(a*)/B(a*) mice showed evidence for hydrocephalus that is always found in B(-)/B(-) mice. Rescue of this defect was due to proper localization and function of GFP-NMHC II-A in place of NMHC II-B in a cell-cell adhesion complex in the cells lining the spinal canal. Restoration of the integrity and adhesion of these cells prevents protrusion of the underlying cells into the spinal canal where they block circulation of the cerebral spinal fluid. However, abnormal migration of facial and pontine neurons found in NMHC II-B mutant and ablated mice persisted in B(a*)/B(a*) mice. Thus, although NMHC II-A can substitute for NMHC II-B to maintain integrity of the spinal canal, NMHC II-B plays an isoform-specific role during cytokinesis in cardiac myocytes and in migration of the facial and pontine neurons.     

Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1-591, delta N592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and delta N592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and delta N592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed delta N592 did not bind to actin, suggesting that delta N592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of delta N592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of delta N592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not delta N592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.     

Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis

RNA interference (RNAi) treatment of monkey COS-7 cells, a cell line that lacks nonmuscle myosin heavy chain II-A (NMHC II-A) but contains NMHC II-B and II-C, was used to investigate the participation of NMHC isoforms in cytokinesis. We specifically suppressed the expression of NMHC II-B or II-C using 21 nucleotide small interfering RNA (siRNA) duplexes. Down-regulation of NMHC II-B protein expression to 10.2 +/- 0.7% inhibited COS-7 cell proliferation by 50% in the RNAi-treated cells compared with control cells. Moreover, whereas 8.7 +/- 1.0% of control cells were multinucleated, 62.4 +/- 8.8% of the NMHC II-B RNAi-treated cells were multinucleated 72 h after transfection. The RNAi-treated cells had increased surface areas and, unlike control cells, lacked actin stress fibers. Treatment of the COS-7 cells with NMHC II-C siRNA decreased NMHC II-C expression to 5.2 +/- 0.1% compared with the endogenous content of II-C; however, down-regulation of NMHC II-C did not cause increased multinucleation. Immunoblot analysis using a pan-myosin antibody showed that the content of NMHC II-C was less than one-twentieth the amount of NMHC II-B, thereby explaining the lack of response to II-C siRNA. Introducing green fluorescent protein (GFP)-tagged NMHC II isoforms into II-B siRNA-treated cells resulted in reduction of multinucleation from 62.4 +/- 8.8% to 17.8 +/- 2.2% using GFP-NMHC II-B, to 29.8 +/- 7.4% using GFP-NMHC II-A, and to 34.1 +/- 8.6% using NMHC II-C-GFP. These studies have shown that expression of endogenous NMHC II-C in COS-7 cells is insufficient for normal cytokinesis and that exogenous NMHC II-A and NMHC II-C can, at least partially, rescue the defect in cytokinesis due to the loss of NMHC II-B.     

A specific isoform of nonmuscle myosin II-C is required for cytokinesis in a tumor cell line

Nonmuscle myosin IIs play an essential role during cytokinesis. Here, we explore the function of an alternatively spliced isoform of nonmuscle myosin heavy chain (NMHC) II-C, called NMHC II-C1, in the A549 human lung tumor cell line during cytokinesis. NMHC II-C1 contains an insert of 8 amino acids in the head region of NMHC II-C. First, we show that there is a marked increase in both the mRNA encoding NMHC II-C1 and protein in tumor cell lines compared with nontumor cell lines derived from the same tissue. Quantification of the amount of myosin II isoforms in the A549 cells shows that the amounts of NMHC II-A and II-C1 protein are about equal and substantially greater than NMHC II-B. Using specific siRNAs to decrease NMHC II-C1 in cultured A549 cells resulted in a 5.5-fold decrease in the number of cells at 120 h, whereas decreasing NMHC II-A with siRNA does not affect cell proliferation. This decreased proliferation can be rescued by reintroducing NMHC II-C1 but not NMHC II-A or II-B into A549 cells, although noninserted NMHC II-C does rescue to a limited extent. Time lapse video microscopy revealed that loss of NMHC II-C1 leads to a delay in cytokinesis and prolongs it from 2 to 8-10 h. These findings are consistent with the localization of NMHC II-C1 to the intercellular bridge that attaches the two dividing cells during the late phases of cytokinesis. The results suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line.     

Disease-associated mutations and alternative splicing alter the enzymatic and motile activity of nonmuscle myosins II-B and II-C

Human families with single amino acid mutations in nonmuscle myosin heavy chain (NMHC) II-A (MYH9) and II-C (MYH14) have been described as have mice generated with a point mutation in NMHC II-B (MYH10). These mutations (R702C and N93K in human NMHC II-A, R709C in murine NMHC II-B, and R726S in human NMHC II-C) result in phenotypes affecting kidneys, platelets, and leukocytes (II-A), heart and brain (II-B), and the inner ear (II-C). To better understand the mechanisms underlying these defects, we characterized the in vitro activity of mutated and wild-type baculovirus-expressed heavy meromyosin (HMM) II-B and II-C. We also expressed two alternatively spliced isoforms of NMHC II-C which differ by inclusion/exclusion of eight amino acids in loop 1, with and without mutations. Comparison of the actin-activated MgATPase activity and in vitro motility shows that mutation of residues Asn-97 and Arg-709 in HMM II-B and the homologous residue Arg-722 (Arg-730 in the alternatively spliced isoform) in HMM II-C decreases both parameters but affects in vitro motility more severely. Analysis of the transient kinetics of the HMM II-B R709C mutant shows an extremely tight affinity of HMM for ADP and a very slow release of ADP from acto-HMM. Although mutations generally decreased HMM activity, the R730S mutation in HMM II-C, unlike the R730C mutation, had no effect on actin-activated MgATPase activity but decreased the rate of in vitro motility by 75% compared with wild type. Insertion of eight amino acids into the HMM II-C heavy chain increases both actin-activated MgATPase activity and in vitro motility.     

Nonmuscle myosin II localizes to the Z-lines and intercalated discs of cardiac muscle and to the Z-lines of skeletal muscle

To understand the role of nonmuscle myosin II in cardiac and skeletal muscle, we used a number of polyclonal antibodies, three detecting nonmuscle myosin heavy chain II-B (NMHC II-B) and two detecting NMHC II-A, to examine the localization of these two proteins in fresh-frozen, acetone-fixed sections of normal human and mouse hearts and human skeletal muscles. Results were similar in both species and were confirmed by examination of fresh-frozen sections of human hearts subjected to no fixation or to treatment with either 4% p-formaldehyde or 50% glycerol. NMHC II-B was diffusely distributed in the cytoplasm of cardiac myocytes during development, but after birth it was localized to the Z-lines and intercalated discs. Dual labeling showed almost complete colocalization of NMHC II-B with alpha-actinin. Whereas endothelial cells, smooth muscle cells and fibroblasts showed strong immunoreactivity for NMHC II-A and NMHC II-B, cardiac myocytes only showed reactivity for the latter. The Z-lines of human skeletal muscle cells, in contrast to those of cardiac myocytes, gave positive reactions for both NMHC II-A and NMHC II-B. The presence of a motor protein in the Z-lines and intercalated discs raises the possibility that these structures may play a more dynamic role in the contraction/relaxation mechanism of cardiac and skeletal muscle than has been previously suspected.     

Identification and characterization of nonmuscle myosin II-C, a new member of the myosin II family

A previously unrecognized nonmuscle myosin II heavy chain (NMHC II), which constitutes a distinct branch of the nonmuscle/smooth muscle myosin II family, has recently been revealed in genome data bases. We characterized the biochemical properties and expression patterns of this myosin. Using nucleotide probes and affinity-purified antibodies, we found that the distribution of NMHC II-C mRNA and protein (MYH14) is widespread in human and mouse organs but is quantitatively and qualitatively distinct from NMHC II-A and II-B. In contrast to NMHC II-A and II-B, the mRNA level in human fetal tissues is substantially lower than in adult tissues. Immunofluorescence microscopy showed distinct patterns of expression for all three NMHC isoforms. NMHC II-C contains an alternatively spliced exon of 24 nucleotides in loop I at a location analogous to where a spliced exon appears in NMHC II-B and in the smooth muscle myosin heavy chain. However, unlike neuron-specific expression of the NMHC II-B insert, the NMHC II-C inserted isoform has widespread tissue distribution. Baculovirus expression of noninserted and inserted NMHC II-C heavy meromyosin (HMM II-C/HMM II-C1) resulted in significant quantities of expressed protein (mg of protein) for HMM II-C1 but not for HMM II-C. Functional characterization of HMM II-C1 by actin-activated MgATPase activity demonstrated a V(max) of 0.55 + 0.18 s(-1), which was half-maximally activated at an actin concentration of 16.5 + 7.2 microm. HMM II-C1 translocated actin filaments at a rate of 0.05 + 0.011 microm/s in the absence of tropomyosin and at 0.072 + 0.019 microm/s in the presence of tropomyosin in an in vitro motility assay.     

A point mutation in the motor domain of nonmuscle myosin II-B impairs migration of distinct groups of neurons

We generated mice harboring a single amino acid mutation in the motor domain of nonmuscle myosin heavy chain II-B (NMHC II-B). Homozygous mutant mice had an abnormal gait and difficulties in maintaining balance. Consistent with their motor defects, the mutant mice displayed an abnormal pattern of cerebellar foliation. Analysis of the brains of homozygous mutant mice showed significant defects in neuronal migration involving granule cells in the cerebellum, the facial neurons, and the anterior extramural precerebellar migratory stream, including the pontine neurons. A high level of NMHC II-B expression in these neurons suggests an important role for this particular isoform during neuronal migration in the developing brain. Increased phosphorylation of the myosin II regulatory light chain in migrating, compared with stationary pontine neurons, supports an active role for myosin II in regulating their migration. These studies demonstrate that NMHC II-B is particularly important for normal migration of distinct groups of neurons during mouse brain development.     

Distinct and redundant roles of the non-muscle myosin II isoforms and functional domains

We propose that the in vivo functions of NM II (non-muscle myosin II) can be divided between those that depend on the N-terminal globular motor domain and those less dependent on motor activity but more dependent on the C-terminal domain. The former, being more dependent on the kinetic properties of NM II to translocate actin filaments, are less amenable to substitution by different NM II isoforms, whereas the in vivo functions of the latter, which involve the structural properties of NM II to cross-link actin filaments, are more amenable to substitution. In light of this hypothesis, we examine the ability of NM II-A, as well as a motor-compromised form of NM II-B, to replace NM II-B and rescue neuroepithelial cell-cell adhesion defects and hydrocephalus in the brain of NM II-B-depleted mice. We also examine the ability of NM II-B as well as chimaeric forms of NM II (II-A head and II-B tail and vice versa) to substitute for NM II-A in cell-cell adhesions in II-A-ablated mice. However, we also show that certain functions, such as neuronal cell migration in the developing brain and vascularization of the mouse embryo and placenta, specifically require NM II-B and II-A respectively.     

Loss of cell adhesion causes hydrocephalus in nonmuscle myosin II-B-ablated and mutated mice

Ablation of nonmuscle myosin (NM) II-B in mice during embryonic development leads to marked enlargement of the cerebral ventricles and destruction of brain tissue, due to hydrocephalus. We have identified a transient mesh-like structure present at the apical border of cells lining the spinal canal of mice during development. This structure, which only contains the II-B isoform of NM, also contains beta-catenin and N-cadherin, consistent with a role in cell adhesion. Ablation of NM II-B or replacement of NM II-B with decreased amounts of a mutant (R709C), motor-impaired NM II-B in mice results in collapse of the mesh-like structure and loss of cell adhesion. This permits the underlying neuroepithelial cells to invade the spinal canal and obstruct cerebral spinal fluid flow. These defects in the CNS of NM II-B-ablated mice seem to be the cause of hydrocephalus. Interestingly, the mesh-like structure and patency of the spinal canal can be restored by increasing expression of the motor-impaired NM II-B, which also rescues hydrocephalus. However, the mutant isoform cannot completely rescue neuronal cell migration. These studies show that the scaffolding properties of NM II-B play an important role in cell adhesion, thereby preventing hydrocephalus during mouse brain development.     

A Highlights from MBoC Selection: Ablation of Nonmuscle Myosin II-B and II-C Reveals a Role for Nonmuscle Myosin II in Cardiac Myocyte Karyokinesis

Ablation of nonmuscle myosin (NM) II-A or NM II-B results in mouse embryonic lethality. Here, we report the results of ablating NM II-C as well as NM II-C/II-B together in mice. NM II-C ablated mice survive to adulthood and show no obvious defects compared with wild-type littermates. However, ablation of NM II-C in mice expressing only 12% of wild-type amounts of NM II-B results in a marked increase in cardiac myocyte hypertrophy compared with the NM II-B hypomorphic mice alone. In addition, these hearts develop interstitial fibrosis associated with diffuse N-cadherin and β-catenin localization at the intercalated discs, where both NM II-B and II-C are normally concentrated. When both NM II-C and II-B are ablated the B⁻C⁻/B⁻C⁻ cardiac myocytes show major defects in karyokinesis. More than 90% of B⁻C⁻/B⁻C⁻ myocytes demonstrate defects in chromatid segregation and mitotic spindle formation accompanied by increased stability of microtubules and abnormal formation of multiple centrosomes. This requirement for NM II in karyokinesis is further demonstrated in the HL-1 cell line derived from mouse atrial myocytes, by using small interfering RNA knockdown of NM II or treatment with the myosin inhibitor blebbistatin. Our study shows that NM II is involved in regulating cardiac myocyte karyokinesis by affecting microtubule dynamics.     

Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A

Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or β1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.     

The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the nonspliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells [X. Ma, S. Kawamoto, J. Uribe, R.S. Adelstein, Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development, Mol. Biol. Cell 15 (2006) 2138-2149]. In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acid II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific.     

Keratin 5-Cre-driven excision of nonmuscle myosin IIA in early embryo trophectoderm leads to placenta defects and embryonic lethality

In studies initially focused on roles of nonmuscle myosin IIA (NMIIA) in the developing mouse epidermis, we have discovered that a previously described cytokeratin 5 (K5)-Cre gene construct is expressed in early embryo development. Mice carrying floxed alleles of the nonmuscle myosin II heavy chain gene (NMHC IIA^flox/flox) were crossed with the K5-Cre line. The progeny of newborn pups did not show a Mendelian genotype distribution, suggesting embryonic lethality. Analysis of post-implantation conceptuses from embryonic day (E)9.5 to E13.5 revealed poorly developed embryos and defective placentas, with significantly reduced labyrinth surface area and blood vessel vascularization. These results suggested the novel possibility that the bovine K5 promoter-driven Cre-recombinase was active early in trophoblast-lineage cells that give rise to the placenta. To test this possibility, K5-Cre transgenic mice were crossed with the mT/mG reporter mouse in which activation of GFP expression indicates Cre transgene expression. We observed activation of K5-Cre-driven GFP expression in the ectoplacental cone, in the extraembryonic ectoderm, and in trophoblast giant cells in the E6.5 embryo. In addition, we observed GFP expression at E11.5 to E13.5 in both the labyrinth of the placenta and the yolk sac. NMIIA expression was detected in these same cell types in normal embryos, as well as in E13.5 yolk sac and labyrinth. These findings taken together suggest that NMHC IIA may play critical roles in the early trophoblast-derived ectoplacental cone and extraembryonic ectoderm, as well as in the yolk sac and labyrinth tissues that form later. Our findings are consistent with phenotypes of constitutive NMIIA knockout mice made earlier, that displayed labyrinth and yolk sac-specific defects, but our findings extend those observations by suggesting possible NMIIA roles in trophoblast lineages as well. These results furthermore demonstrate that K5-Cre gene constructs, previously reported to be activated starting at approximately E12.5 in the forming epidermis, may be widely useful as drivers for activation of cre/lox based gene excision in early embryo extraembronic trophoblast tissues as well.     

Correlation between the clinical phenotype of MYH9-related disease and tissue distribution of class II nonmuscle myosin heavy chains

Nonmuscle myosin heavy chain II-A is responsible for MYH9-related disease, which is characterized by macrothrombocytopenia, granulocyte inclusions, deafness, cataracts, and renal failure. Since another two highly conserved nonmuscle myosins, II-B and II-C, are known, an analysis of their tissue distribution is fundamental for the understanding of their biological roles. In mouse, we found that all forms are ubiquitously expressed. However, megakaryocytic and granulocytic lineages express only II-A, suggesting that congenital features, macrothrombocytopenia, and leukocyte inclusions correlate with its exclusive presence. In kidney, eye, and ear, where clinical manifestations have a late onset, as well as in other tissues apparently not affected in patients, II-A and at least one of the other two isoforms are expressed, suggesting that II-B and II-C can partially compensate for each other. We hypothesize that cells expressing only II-A manifest the congenital defects, while tissues expressing additional myosin II isoforms show either late onset of abnormalities or no pathological sign.     

Increased expression of nonmuscle myosin IIs is associated with 3MC-induced mouse tumor

Administration of the chemical carcinogen, 3-methylcholanthrene (3MC), in the hind leg induces the progressive formation of tumors in mice within 110 days. Previous reports suggest that transformation of muscle cells to atypical cells is one of the causes of tumor formation. Molecular events that lead to transformation of normal cells to atypical cells are not well understood. Here, we investigate the effect of 3MC on the expression of nonmuscle myosin IIs (NM IIs) which are known to be involved in cell migration, division and adhesion. Mass spectroscopy analysis reveals that tumor tissue contains 64.5% NM II-A, 34% II-B and only 1.5% II-C of total NM IIs, whereas these three isoforms of NM IIs are undetectable by mass spectroscopy in normal tissue associated with the tumor (NTAT) from the hind leg. Quantification of heavy chain mRNAs of NM II suggests that tumor tissue contains 25.7-fold and 19.03-fold more of NM II-A and II-B, respectively, compared with NTAT. Unlike NM II-B, which is detected only after tumor formation, II-A is detectable as early as day 7 after a second dose of 3MC. Immunofluorescence confocal microscopy reveals that fibroblast cells which are sparsely distributed in normal tissue are densely populated but of atypical shape in the tumor. These findings suggest that transformation of fibroblasts or non-fibroblast cells to atypical, cancerous cells is associated with increased levels of NM II-A and NM II-B expression in the 3MC-induced tumor mouse model. 3MC-induced transformation is further demonstrated in C2C12 myotubes.     

Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development

We report that the alternatively spliced isoforms of nonmuscle myosin heavy chain II-B (NHMC II-B) play distinct roles during mouse brain development. The B1-inserted isoform of NMHC II-B, which contains an insert of 10 amino acids near the ATP-binding region (loop 1) of the myosin heavy chain, is involved in normal migration of facial neurons. In contrast, the B2-inserted isoform, which contains an insert of 21 amino acids near the actin-binding region (loop 2), is important for postnatal development of cerebellar Purkinje cells. Deletion of the B1 alternative exon, together with reduced expression of myosin II-B, results in abnormal migration and consequent protrusion of facial neurons into the fourth ventricle. This protrusion is associated with the development of hydrocephalus. Restoring the amount of myosin II-B expression to wild-type levels prevents these defects, showing the importance of total myosin activity in facial neuron migration. In contrast, deletion of the B2 alternative exon results in abnormal development of cerebellar Purkinje cells. Cells lacking the B2-inserted isoform show reduced numbers of dendritic spines and branches. Some of the B2-ablated Purkinje cells are misplaced in the cerebellar molecular layer. All of the B2-ablated mice demonstrated impaired motor coordination.     

Epidermal growth factor-mediated transient phosphorylation and membrane localization of myosin II-B are required for efficient chemotaxis

Epidermal growth factor (EGF) stimulation of prostate metastatic tumor cells results in transient phosphorylation and cellular localization of non-muscle myosin heavy chain II-B (NMHC II-B) with kinetics similar to those seen in chemotaxis. We demonstrate that expression of 18- and 72-kDa fragments derived from the NMHC II-B C terminus that contain EGF-dependent NMHC II-B phosphorylation sites serve as dominant-negative mutations for EGF-dependent NMHC II-B phosphorylation and localization. Both fragments inhibited the EGF-dependent phosphorylation by competing with NMHC II-B on the myosin heavy chain kinase. However, only expression of the 72-kDa fragment resulted in cells with abnormalities in cell shape, focal adhesions, and chemotaxis. We found that the 72-kDa (but not 18-kDa) fragment is capable of self-assembly. To our knowledge, these results provide the first strong evidence that EGF-dependent NMHC II-B phosphorylation is required for the cellular localization of NMHC II-B and that NMHC II-B is required for normal cell attachment and for chemotactic response.     

Basic mechanism of three-dimensional collagen fibre transport by fibroblasts

Collagen remodelling by fibroblasts has a crucial role in organizing tissue structures that are essential to motility during wound repair, development and regulation of cell growth. However, the mechanism of collagen fibre movement in three-dimensional (3D) matrices is not understood. Here, we show that fibroblast lamellipodia extend along held collagen fibres, bind, and retract them in a 'hand-over-hand' cycle, involving alpha2beta1 integrin. Wild-type fibroblasts move collagen fibres three to four times farther per cycle than fibroblasts lacking myosin II-B (myosin II-B(-/-)). Similarly, myosin II-B(-/-) fibroblasts contract 3D collagen gels threefold less than controls. On two-dimensional (2D) substrates, however, rates of collagen bead and cell movement are not affected by loss of myosin II-B. Green fluorescent protein (GFP)-tagged myosin II-B, but not II-A, restores normal function in knockout cells and localizes to cell processes, whereas myosin II-A is more centrally located. Additionally, GFP-myosin II-B moves out to the periphery and back during hand-over-hand fibre movement, whereas on 2D collagen, myosin II-B is more centrally distributed. Thus, we suggest that cyclic myosin II-B assembly and contraction in lamellipodia power 3D fibre movements.     

Induction of nonmuscle myosin heavy chain II-C by butyrate in RAW 264.7 mouse macrophages

RAW 264.7 macrophages express nonmuscle myosin heavy chain II-A as the only significant nonmuscle myosin heavy chain isoform, with expression of nonmuscle myosin heavy chain II-B and II-C low or absent. Treatment of the cells with sodium butyrate, an inhibitor of histone deacetylase, led to the dose-dependent induction of nonmuscle myosin heavy chain II-C. Trichostatin A, another inhibitor of histone deacetylase, also induced nonmuscle myosin heavy chain II-C. Induction of nonmuscle myosin heavy chain II-C in response to these histone deacetylase inhibitors was attenuated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. Bacterial lipopolysaccharide alone had no effect on basal nonmuscle myosin heavy chain II-C expression, but attenuated butyrate-mediated induction of nonmuscle myosin heavy chain II-C. The effects of lipopolysaccharide were mimicked by the nitric oxide donors sodium nitroprusside and spermine NONOate, suggesting a role for nitric oxide in the lipopolysaccharide-mediated down-regulation of nonmuscle myosin heavy chain II-C induction. This was supported by experiments with the inducible nitric-oxide synthase inhibitor 1400W, which partially blocked the lipopolysaccharide-mediated attenuation of nonmuscle myosin heavy chain induction. 8-Bromo-cGMP had no effect on nonmuscle myosin heavy chain induction, consistent with a cGMP-independent mechanism for nitric oxide-mediated inhibition of nonmuscle myosin heavy chain II-C induction.