Electron microscopic localization of cytoplasmic myosin with ferritin- labeled antibodies

We localized myosin in vertebrate nonmuscle cells by electron microscopy using purified antibodies coupled with ferritin. Native and formaldehyde-fixed filaments of purified platelet myosin filaments each consisting of approximately 30 myosin molecules bound an equivalent number of ferritin-antimyosin conjugates. In preparations of crude platelet actomyosin, the ferritin-antimyosin bound exclusively to similar short, 10-15 nm wide filaments. In both cases, binding of the ferritin-antimyosin to the myosin filaments was blocked by preincubation with unlabeled antimyosin. With indirect fluorescent antibody staining at the light microscope level, we found that the ferritin-antimyosin and unlabeled antimyosin stained HeLa cells identically, with the antibodies concentrated in 0.5-microns spots along stress fibers. By electron microscopy, we found that the concentration of ferritin-antimyosin in the dense regions of stress fibers was five to six times that in the intervening less dense regions, 20 times that in the cytoplasmic matrix, and 100 times that in the nucleus. These concentration differences may account for the light microscope antibody staining pattern of spread interphase cells. Some, but certainly not all, of the ferritin-antimyosin was associated with 10-15-nm filaments. In mouse intestinal epithelial cells, ferritin- antimyosin was located almost exclusively in the terminal web. In isolated brush borders exposed to 5 mM MgCl2, ferritin-antimyosin was also concentrated in the terminal web associated with 10-15-nm filaments.     

Nonmuscle myosin II localization is regulated by JNK during Drosophila larval wound healing

We investigated cell shape changes during wound closure in the Drosophila larval epidermis. During reepithelialization, epidermal cells permanently change shape from pentagonal or hexagonal to irregular forms. This process requires zipper, a gene encoding the Drosophila nonmuscle myosin II heavy chain. Following wounding, myosin II is localized at the wound margin and at the rear end of individual cells located within several rows from the wound hole. The c-Jun N-terminal kinase (JNK) pathway is essential for this myosin II localization. These results suggest that not only the wound leading edge but also the cells lying distal to the leading edge cells actively participate in epithelial cell sheet migration during wound hole closure.     

Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II paralogs A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modeling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between paralogs A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-localizes with pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two paralogs form a balanced system for regulated force generation.     

Nonmuscle and smooth muscle myosin isoforms in bovine endothelial cells

A panel of monoclonal antibodies, specific for human platelet (NM-A9, NM-F6, and NM-G2) and for bovine smooth muscle (SM-E7) myosin heavy chains (MHC), were used to study the composition and the distribution of myosin isoforms in bovine endothelial cells (EC), in vivo and in vitro. Using indirect and double immunofluorescence techniques, we have found that in the intact aortic endothelium there is expression of nonmuscle MHC (NM-MHC), exclusively. By contrast, hepatic sinusoidal endothelium as well as cultured bovine aortic EC (BAEC) in the subconfluent phase of growth show coexistence of NM- and smooth muscle MHC (SM-MHC) isoforms. SM myosin immunoreactivity disappears when cultured BAEC become confluent. In this phase of cell growth, NM-MHC isoforms are localized differently within the cells, i.e., in the cytoplasm around the nucleus or in the cortical, submembranous region of EC cytoplasm. A third type of intracellular distribution of NM-MHC immunoreactivity was evident in the cell periphery of binucleated, confluent BAEC. These data indicate that (1) several myosin isoforms are differently distributed in bovine endothelia; and (2) SM myosin expression and the specific subcellular localization of NM myosin isoforms within EC might be regulated by cell-cell interactions.     

Nonmuscle myosin phosphorylation sites for calcium-dependent and calcium-independent protein kinases

Thymus myosin, light chains and a synthetic peptide (S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S-N-V-F-S) corresponding to the N-terminal sequence of smooth muscle myosin light chains were compared as substrates for calcium/calmodulin-dependent protein kinase (MLCK), calcium/phospholipid-dependent protein kinase (PKC), and a MgATP-activated protein kinase (H4PK) from lymphoid cells. All protein kinases catalyzed phosphorylation of the substrates although H4PK showed higher affinity for isolated light chains and the peptide. Phosphoamino acid analysis and analysis of thermolysin peptides established that PKC catalyzed phosphorylation of threonine-9 or 10. In addition, PKC and H4PK catalyzed phosphorylation at serine-19, the MLCK site. Collectively the data support the hypothesis that myosin filament assembly in nonmuscle cells may be regulated by a variety of calcium-dependent and calcium-independent protein kinases.     

Nonmuscle myosin IIb is involved in the guidance of fibroblast migration

Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.     

Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow

Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that approximately 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and approximately 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.     

Nonmuscle myosin IIB, a sarcomeric component in the extraocular muscles

Extraocular muscles (EOMs) are categorized as skeletal muscles; however, emerging evidence indicates that their gene expression profile, metabolic characteristics and functional properties are significantly different from the prototypical members of this muscle class. Gene expression profiling of developing and adult EOM suggest that many myofilament and cytoskeletal proteins have unique expression patterns in EOMs, including the maintained expression of embryonic and fetal isoforms of myosin heavy chains (MyHC), the presence of a unique EOM specific MyHC and mixtures of both cardiac and skeletal muscle isoforms of thick and thin filament accessory proteins. We demonstrate that nonmuscle myosin IIB (nmMyH IIB) is a sarcomeric component in ∼ 20% of the global layer fibers in adult rat EOMs. Comparisons of the myofibrillar distribution of nmMyHC IIB with sarcomeric MyHCs indicate that nmMyH IIB co-exists with slow MyHC isoforms. In longitudinal sections of adult rat EOM, nmMyHC IIB appears to be restricted to the A-bands. Although nmMyHC IIB has been previously identified as a component of skeletal and cardiac sarcomeres at the level of the Z-line, the novel distribution of this protein within the A band in EOMs is further evidence of both the EOMs complexity and unconventional phenotype.     

Nonmuscle myosin II-B (myh10) expression analysis during zebrafish embryonic development

Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes (myh9, myh10, and myh14) encoding different nonmuscle myosin heavy chains. The three NM II isoforms share a very similar molecular structure and play important roles in a variety of fundamental biological processes. NM II-B (myh10) has been shown to be essential for the formation of mouse neural system and heart. But so far the complete knowledge for its expression in developing zebrafish embryos is lacking. In current study, we proved the conservation of zebrafish NM II-B in vertebrate evolution by in silicon analysis. Afterwards the NM II-B (myh10) expression was demonstrated to initiate after gastrulation stage. At 20 hpf, the expression is mainly restricted in central nervous system (CNS). It was maintained and expanded to sensor organ including eye, otic vesicle, and olfactory bulb at 36 hpf and later. We also detected myh10 mRNA hybridization signal in 48 hpf zebrafish heart. In addition, we investigated myh9a and myh9b mRNA distribution in zebrafish developing embryos. It was shown that myh10 and myh9 have distinct expression pattern, with myh9s not in neural system but in epidermis, enveloping layer (EVL). Our study provides new insight into the NM II expression and the use of this model organism to tackle future studies on the role of NM II in embryo development.     

Pig brain homogenates contain smooth muscle myosin and cytoplasmic myosin isoforms

Summary Three myosin isoforms, two of smooth muscle and one of cytoplasmic origin, were found in porcine brain by Western blotting analysis with antibodies specific for smooth and cytoplasmic myosins. The smooth muscle isoforms comprise at least 30% of the total myosin present. Brain tissue is therefore not a suitable source for the isolation of pure cytoplasmic contractile proteins.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - FITC fluorescein isothiocyanate - PBS phosphate buffered saline - SDS PAGE polyacrylamide electrophoresis in the presence of sodium dodecylsulphate - TRIS tris(hydroxymethyl)aminomethane     

Chara myosin and the energy of cytoplasmic streaming

Recently, it was found that myosin generating very fast cytoplasmic streaming in Chara corallina has very high ATPase activity. To estimate the energy consumed by this myosin, its concentration in the internodal cells of C. corallina was determined by quantitative immunoblot. It was found that the concentration of Chara myosin was considerably high (200 nM) and the amount of ATP consumed by this myosin would exceed that supplied by dark respiration if all myosin molecules were fully activated by the interaction with actin. These results and model calculations suggested that the energy required to generate cytoplasmic streaming is very small and only one-hundredth of the existing myosin is enough to maintain the force for the streaming in the Chara cell.     

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca²⁺ dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity. actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions     

A re-evaluation of cytoplasmic gelsolin localization

Gelsolin is a 90,000-mol-wt Ca2+-binding, actin-associated protein that can nucleate actin filament growth, sever filaments, and cap barbed filament ends. Brevin is a closely related 92,000-mol-wt plasma protein with similar properties. Gelsolin has been reported to be localized on actin filaments in stress fibers, in cardiac and skeletal muscle I-bands, and in cellular regions where actin filaments are known to be concentrated. Previous localization studies have used sera or antibody preparations that contain brevin. Using purified brevin-free IgG and IgA monoclonal antibodies or affinity-purified polyclonal antibodies for gelsolin and brevin, we find no preferential stress fiber staining in cultured human fibroblasts or I-band staining in isolated rabbit skeletal muscle sarcomeres. Cardiac muscle frozen sections show no pronounced I-band staining, except in local areas where brevin may have penetrated from adjacent blood vessels. Spreading platelets show endogenous gelsolin localized at the cell periphery, in the central cytoplasmic mass and on thin fibers that radiate from the central cytoplasm. Addition of 3-30 micrograms/ml of brevin to the antibodies restores intense stress fiber and I-band staining. We see no evidence for large-scale severing and removal of filaments in stress fibers in formaldehyde-fixed, acetone-permeabilized cells even at brevin concentrations of 30 micrograms/ml. The added brevin or brevin antibody complex binds to actin filaments and is detected by the fluorescently tagged secondary antibody. Brevin binding occurs in either Ca2+ or EGTA, but is slightly more intense in EGTA suggesting some severing and filament removal may occur in Ca2+. The I-band staining is limited to the region where actin and myosin do not overlap. In addition, brevin does not appear to bind at the Z-line. A comparison of cells double-labeled with fluorescein-phallotoxin, exogenous brevin, and a monoclonal antibody, detected with a rhodamine-labeled secondary antibody, shows almost complete co-localization of F-actin with the brevin-gelsolin-binding sites. A major exception is in the area of the adhesion plaque. A quantitative comparison of the fluorescein-rhodamine fluorescence intensities along a stress fiber and into the adhesion plaque shows that the fluorescein signal, associated with F-actin, increases while the rhodamine signal decreases. We infer that exogenous brevin or endogenous gelsolin can bind to and potentially sever most actin filaments, but that actin-associated proteins in the adhesion plaque can prevent binding and severing.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anillin promotes astral microtubule-directed cortical myosin polarization

Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle-directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex.     

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.     

Differential localization of Acanthamoeba myosin I isoforms

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.