Differential localization of cytoplasmic myosin ii isoforms a and b in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro

We have isolated a novel, high M r protein from human retinal pigment epithelial cells and endothelial cells by affinity chromatography on Sepharose 4B. Two polypeptides are present on SDS-gels of the 8 M urea eluent with apparent molecular mass of ϳ210 and 47 kDa. In the absence of dithiothreitol, the two polypeptides migrate as one protein band with an apparent molecular mass of ϳ550 kDa. "Piglet," as this molecule is tentatively named, is present in retinal pigment epithelial and endothelial cells of several species, but could not be detected in the nonepithelial cells we examined. Immunofluorescent localization using an antibody to the 210-kDa polypeptide revealed a filamentous network in the cytoplasm of cultured cells. This antibody was used to identify a cDNA for piglet in a bovine aortic endothelial cell expression library. Sequence data indicate a high degree of identity with non-muscle myosin II heavy chain. We subsequently found that piglet had an actinactivated ATPase activity, colocalized with actin in cells, and reacted on Western blots with a pan-non-muscle myosin II heavy chain antiserum. The protein was also recognized by antibodies specific for myosin heavy chain isoform A, but did not react with anti-isoform B antibodies. Although piglet has several features in common with known forms of non-muscle myosin II, the distinctly unconventional features it displays suggest that it is a novel myosin.The retinal pigment epithelium (RPE) 1 is composed of a population of cuboidal to low columnar cells sandwiched between the neural retina and the highly vascularized choroid layer of the eye. Although RPE cells are normally nonproliferative and nonmigratory, they are highly active in other respects. They are indespensible for the function and survival of their immediate neighbors, the rods and cones, and malfunctions in the RPE form the basis for a variety of blinding disorders.The RPE cell feature that is perhaps most directly related to vision is the presence of dense pigment granules in their cytoplasm. These granules absorb light that has passed through the neural layers of the retina, preventing backscatter and unwanted reflection. The RPE also performs a variety of metabolic functions in support of the neural retina. By means of tight junctions, they create the blood-ocular barrier that regulates the passage of molecules between the retina and choroidal capillaries (1). They are, therefore, primary determinants of the photoreceptor microenvironment. The RPE is also responsible for phagocytosis and disposal of shed photoreceptor outer segments (2). In the absence of this function, degeneration of the neural retina ensues. Moreover, RPE are essential for production of the visual pigment rhodopsin (3). During the visual cycle, the 11-cis-retinaldehyde chromophore of rhodopsin present in photoreceptor cells is reduced to all-trans-retinol. To regenerate rhodopsin, the spent product is transferred to the RPE where it is isomerized to the 11-cis form once again. Directly and indirectly, therefore, the R...

Section: Figmentioning

confidence: 99%

Novel Characteristics of a Myosin Isolated from Mammalian Retinal Pigment Epithelial and Endothelial Cells

Alliegro

Linz

1997

“…Although all class II molecules are composed of two heavy chains, two essential light chains, and two regulatory chains, their unique activities are a function of their particular heavy chain isoforms. Although the nonmuscle heavy chain isoforms share extensive structural homology, they have been shown to demonstrate distinct patterns of expression (15)(16)(17)(18), enzyme kinetics and activation (12, 19 -21), and cellular function (22)(23)(24). Knock-out of either myosin IIA or IIB results in embryonic lethality, although death derives from defects unique to each isoform (25,26).…”

mentioning

confidence: 99%

Regulated Proteolysis of Nonmuscle Myosin IIA Stimulates Osteoclast Fusion

McMichael

Wysolmerski

Lee

2009

The nonmuscle myosin IIA heavy chain (Myh9) is strongly associated with adhesion structures of osteoclasts. In this study, we demonstrate that during osteoclastogenesis, myosin IIA heavy chain levels are temporarily suppressed, an event that stimulates the onset of cell fusion. This suppression is not mediated by changes in mRNA or translational levels but instead is due to a temporary increase in the rate of myosin IIA degradation. Intracellular activity of cathepsin B is significantly enhanced at the onset of osteoclast precursor fusion, and specific inhibition of its activity prevents myosin IIA degradation. Further, treatment of normal cells with cathepsin B inhibitors during the differentiation process reduces cell fusion and bone resorption capacity, whereas overexpression of cathepsin B enhances fusion. Ongoing suppression of the myosin IIA heavy chain via RNA interference results in formation of large osteoclasts with significantly increased numbers of nuclei, whereas overexpression of myosin IIA results in less osteoclast fusion. Increased multinucleation caused by myosin IIA suppression does not require RANKL. Further, knockdown of myosin IIA enhances cell spreading and lessens motility. These data taken together strongly suggest that base-line expression of nonmuscle myosin IIA inhibits osteoclast precursor fusion and that a temporary, cathepsin B-mediated decrease in myosin IIA levels triggers precursor fusion during osteoclastogenesis.The final stages of osteoclastogenesis involve fusion of differentiated precursors from the monocyte/macrophage lineage (1). Although the membrane structural components regulating preosteoclast fusion are not well understood, in recent years a number of candidate cell surface molecules have been implicated, including receptors CD44 (2, 3), CD47 and its ligand macrophage fusion receptor (also known as signal regulatory protein ␣) (4 -6), the purinergic receptor P2X 7 (7), and the disintegrin and metalloproteinase ADAM8 (8). A recently identified receptor, the dendritic cell-specific transmembrane protein, is essential for osteoclast fusion both in vitro and in vivo (9, 10). More recently, the d2 subunit of proton-translocating vacuolar proton-translocating ATPases, a membrane subunit isoform expressed predominantly in osteoclasts, similarly was demonstrated to be required for fusion in vitro and in vivo (11). However, elucidation of the mechanisms by which these molecules may mediate cell fusion has proved to be difficult.The mammalian class II myosin family consists of distinct isoforms expressed in skeletal, smooth, and cardiac muscle, as well as three nonmuscle forms designated IIA, IIB,. Although all class II molecules are composed of two heavy chains, two essential light chains, and two regulatory chains, their unique activities are a function of their particular heavy chain isoforms. Although the nonmuscle heavy chain isoforms share extensive structural homology, they have been shown to demonstrate distinct patterns of expression (15-18), enzyme kinetics and activ...

“…Myosin II is a prominent actor in this context. Myosin II is localized along with actin fibers in the protrusive anterior region and at posterior regions of motile cells, where it is thought to generate contractility, in organizing and breaking cell-substratum adhesion, and/or in reorganizing the actin cytoskeleton (8,9). A recent report finds EGF to induce myosin II heavy chain phosphorylation (at least indirectly), with implications for subcellular localization of the active motor and consequent chemotactic cell movement (10).…”

mentioning

confidence: 99%

Epidermal Growth Factor Induces Fibroblast Contractility and Motility via a Protein Kinase C δ-dependent Pathway

Iwabu

Smith

Allen

et al. 2004

130

115

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKC␦ is the most strongly phosphorylated isoform, and the preferential PKC␦ inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKC␦ is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKC␦ activation, whereas lack of PLC␥ signaling resulted in delayed PKC␦ activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKC␦ is required, a dominant-negative PKC␦ construct or RNAi-mediated PKC␦ depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKC␦ activation. All of the interventions that blocked PKC␦ activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKC␦ activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKC␦ to promote the acute phase of EGF-induced MLC activation.Cell motility induced by activation of epidermal growth factor receptor (EGFR), 1 and related receptor tyrosine kinases, can be deconstructed into a series of orchestrated events: lamellipodial extension, formation of forward adhesions, exertion of contractile forces to pull the cell body forward, and detachment of the rear (1). While each process is required for net cell locomotion, it is not necessarily the case that signals downstream of receptor activation must concomitantly be involved in triggering all of the processes. Despite longstanding anecdotal indications, only recently have formal demonstrations emerged that signaling via EGFR actually elicits cell contractile force generation (2, 3) along with the other biophysical processes (4 -6). Because of the central importance of growth factor-induced cell motility in physiological and pathological applications, such as organogenesis, wound repair, and tumor invasion, determination of key pathways involved in connecting EGFR activity to contractile force generation, as well as the other processes underlying motility, is a crucial undertaking. Myosin motors operating on cytoskeletal actin filaments are presumed to be involved in growth factor-induced cell motility in manne...