The myo2 gene product identified in this study showed similarity to the type II myosin heavy chain in a number of structural features. In addition, Myo2p colocalized with actin overlying mitotic nuclei and could be seen on a shrinking contractile ring. These results strongly suggest that Myo2p composes S. pombe type II myosin, which interacts with actin in the contractile ring and generates force for cytokinesis.. Type II myosin heavy chains known to date carry two IQ motifs, agreeing with the hexameric structure of myosin II. However, we found only one perfect IQ motif (IQXXXRGXXXR) at residues 765-775 in S. pombe Myo2p. A similar sequence to the IQ motif (LQANLQVYNEFR) exists at residues 791-802, but this sequence does not have the central arginine, which is believed to be critical for the binding to the myosin light chain. Thus, we suspect that the type II myosin heavy chain of S. pombe may interact with only one myosin light chain. Interestingly, McCollum et al. (27) observed in their ...

Focal adhesions (FAs) are mechanosensitive adhesion and signaling complexes that grow and change composition in response to myosin II-mediated cytoskeletal tension in a process known as FA maturation. To understand tension-mediated FA maturation, we sought to identify proteins that are recruited to FAs in a myosin II-dependent manner and to examine the mechanism for their myosin II-sensitive FA association. We find that FA recruitment of both the cytoskeletal adapter protein vinculin and the tyrosine kinase FA kinase (FAK) are myosin II and extracellular matrix (ECM) stiffness dependent. Myosin II activity promotes FAK/Src-mediated phosphorylation of paxillin on tyrosines 31 and 118 and vinculin association with paxillin. We show that phosphomimic mutations of paxillin can specifically induce the recruitment of vinculin to adhesions independent of myosin II activity. These results reveal an important role for paxillin in adhesion mechanosensing via myosin II-mediated FAK phosphorylation of ...

Deuterium nuclear magnetic resonance spectroscopy (2H NMR) has been employed to investigate the interaction of lung type II myosin protein with neutral bilayers containing dimyristoylphosphatidylcholine (DMPC) as the only constituent and mixed bilayers containing the negatively charged lipid dimyristoylphosphatidylglycerol (DMPG). DMPC was deuterated at its headgroup by substituting the four protons at the alpha- and beta-positions (DMPC-d4) and the nine protons at the gamma-position (DMPC-d9). DMPG was perdeuterated at its headgroup (DMPG-d5). No changes were observed in the quadrupole splittings or spin-lattice relaxation times for the deuterated DMPC headgroup segments when increasing amounts of myosin were added to liposomes, made exclusively of DMPC-d9 or of DMPC-d4. However, upon the insertion of the negatively charged lipid DMPG at 1:1 molar ratio into the DMPC bilayers, myosin was found to interact electrostatically with the liposomes, thereby affecting significantly both the quadrupole

Phosphorylation of the regulatory light chain of myosin II (MLC(20)) at the activation sites promotes both the motor activity and the filament formation of myosin II, thus playing an important role in various cell motile processes. In contrast, the physiological function of phosphorylation of MLC(20) at the inhibitory sites is unknown. Here we report for the first time the function of the inhibitory site phosphorylation in the cells. We successfully produced the antibodies specifically recognizing the phosphorylation sites of MLC(20) at Ser1, and the platelet-derived growth factor (PDGF)-induced change in the phosphorylation at the Ser1 was monitored. The phosphorylation of MLC(20) at the Ser1 significantly increased during the PDGF-induced actin cytoskeletal reorganization. PDGF disassembled the stress fibers, and this was attenuated with the expression of unphosphorylatable MLC(20) at the Ser1/Ser2 phosphorylation sites. The present results suggest that the down-regulation of myosin II activity

Among a plethora of known effectors of Rho proteins, four recently identified proteins were shown to be required for cytokinesis (Fig. 2). The formin-homology proteins, Drosophila Diaphanous (Castrillon and Wasserman 1994; Wasserman 1998) and its mouse homologue p140mDia1 (Watanabe et al. 1997), bind to and regulate profilin, an actin-binding protein that promotes F-actin polymerization and is required for cytokinesis (Giansanti et al. 1998; Suetsugu et al. 1999). Dictyostelium p21-activated serine/threonine kinase PAKa, a putative Cdc42/Rac effector, is thought to regulate myosin II assembly by inhibiting myosin II heavy chain kinase (Chung and Firtel 1999). Bovine Rho-associated kinase (cleavage furrow kinase) is required for the regulation of the contractile ring contractility and for phosphorylation of intermediate filaments, leading to their disassembly and segregation into daughter cells, which, in turn, ensure efficient cell separation (Kosako et al. 1997, Kosako et al. 1999; Yasui et al. ...

In diseases such as Multiple Sclerosis in the central (CNS) and Guilain-Barre Syndrome in the peripheral (PNS) nervous system, loss of myelin results in conduct...

In diseases such as Multiple Sclerosis in the central (CNS) and Guilain-Barre Syndrome in the peripheral (PNS) nervous system, loss of myelin results in conduct...

This gene encodes a bipartite protein with distinct amino- and carboxy-terminal domains. The amino-terminus contains nuclear localization signals and the carboxy-terminus contains numerous consecutive sequences with extensive similarity to proteins in the gelsolin family of actin-binding proteins, which cap, nucleate, and/or sever actin filaments. The gene product is tightly associated with both actin filaments and plasma membranes, suggesting a role as a high-affinity link between the actin cytoskeleton and the membrane. The encoded protein appears to aid in both myosin II assembly during cell spreading and disassembly of focal adhesions. Several transcript variants encoding different isoforms of supervillin have been described. [provided by RefSeq, Apr 2016 ...

FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a bipartite protein with distinct amino- and carboxy-terminal domains. The amino-terminus contains nuclear localization signals and the carboxy-terminus contains numerous consecutive sequences with extensive similarity to proteins in the gelsolin family of actin-binding proteins, which cap, nucleate, and/or sever actin filaments. The gene product is tightly associated with both actin filaments and plasma membranes, suggesting a role as a high-affinity link between the actin cytoskeleton and the membrane. The encoded protein appears to aid in both myosin II assembly during cell spreading and disassembly of focal adhesions. Several transcript variants encoding different isoforms of supervillin have been described. [provided by RefSeq, Apr 2016 ...

FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a bipartite protein with distinct amino- and carboxy-terminal domains. The amino-terminus contains nuclear localization signals and the carboxy-terminus contains numerous consecutive sequences with extensive similarity to proteins in the gelsolin family of actin-binding proteins, which cap, nucleate, and/or sever actin filaments. The gene product is tightly associated with both actin filaments and plasma membranes, suggesting a role as a high-affinity link between the actin cytoskeleton and the membrane. The encoded protein appears to aid in both myosin II assembly during cell spreading and disassembly of focal adhesions. Several transcript variants encoding different isoforms of supervillin have been described. [provided by RefSeq, Apr 2016 ...

GTPases of the Rho family regulate actinomyosin-based contraction in non-muscle cells. Activation of Rho increases contractility, leading to cell rounding and neurite retraction in neuronal cell lines. Activation of Rac promotes cell spreading and interferes with Rho-mediated cell rounding. Activation of Rac may antagonize Rho by regulating phosphorylation of the myosin-II heavy chain. Stimulation of PC12 cells or N1E-115 neuroblastoma cells with bradykinin induces phosphorylation of threonine residues in the myosin-II heavy chain; this phosphorylation is Ca2+ dependent and regulated by Rac. Both bradykinin-mediated and constitutive activation of Rac promote cell spreading, accompanied by a loss of cortical myosin II. These results identify the myosin-II heavy chain as a new target of Rac-regulated kinase pathways, and implicate Rac as a Rho antagonist during myosin-II-dependent cell-shape changes (van Leeuwen, 1999). The molecular events responsible for Rac-mediated cytoskeletal changes are not ...

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Throughout development, tissues exhibit dynamic cell deformation, which is characterized by the integration of cell boundary contraction and/or elongation. Such changes ultimately establish tissue morphology and function. In comparison to cell boundary contraction, which is predominantly driven by non-muscle myosin II (MyoII)-dependent contraction, the mechanisms of cell boundary elongation remain elusive. This study explored the dynamics of the amnioserosa, which is known to exhibit cell shape oscillation, as a model system to study the subcellular-level mechanics that spatiotemporally evolve during Drosophila dorsal closure. Cell boundary elongation is shown to occur through a combination of a non-autonomous active process and an autonomous process. The former is driven by a transient change in the level of MyoII in the neighboring cells that pull the vertices, whereas the latter is governed by the relaxation of junctional tension. By monitoring cell boundary deformation during live imaging, ...

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Despite their relevance in sculpting functional embryonic structures, very little is known about the mechanisms by which cellular forces affect/control developmental processes, mainly because it has not been possible to measure cellular forces within developing tissues in vivo. In this talk, I will present a new technique that permits direct quantification of cellular mechanical stresses in situ within living tissues and developing organs. Using this novel technique, we quantify the stresses generated by mammary epithelial cells cultured within 3D aggregates (3.4 nN/µm2) and confirm that these stresses are dependent on myosin II activity and more than two-fold larger than the stresses generated by cells of embryonic tooth mesenchyme when analyzed within similar cultured aggregates or in developing whole mouse mandibles.. ...

King-Smith, Christina, and Ivraym B. Barsoum. Blebbistatin, a Myosin II Inhibitor, Blocks Melanosome Aggregation But Not Dispersion in Fish Retinal Pigment Epithelial (RPE) Cells. Molecular Biology of the Cell 14 Supp. (2003). Abstract.. ...

During cell migration, myosin II modulates adhesion, cell protrusion and actin organization at the leading edge. We show that an F-actin- and membrane-associated scaffolding protein, called supervillin (SV, p205), binds directly to the subfragment 2 domains of nonmuscle myosin IIA and myosin IIB and to the N-terminus of the long form of myosin light chain kinase (L-MLCK). SV inhibits cell spreading via an MLCK- and myosin II-dependent mechanism. Overexpression of SV reduces the rate of cell spreading, and RNAi-mediated knockdown of endogenous SV increases it. Endogenous and EGFP-tagged SV colocalize with, and enhance the formation of, cortical bundles of F-actin and activated myosin II during early cell spreading. The effects of SV are reversed by inhibition of myosin heavy chain (MHC) ATPase (blebbistatin), MLCK (ML-7) or MEK (U0126), but not by inhibiting Rho-kinase with Y-27632. Flag-tagged L-MLCK co-localizes in cortical bundles with EGFP-SV, and kinase-dead L-MLCK disorganizes these bundles. The L

Although cytoskeletal proteins such as myosin II are implicated in the control of insulin secretion, their precise role is poorly understood. In other secretory cells, myosin II is predominantly regulated via the phosphorylation of the regulatory light chains (RLC). The current study was aimed at investigating RLC phosphorylation in beta-cells. In both the insulin-secreting cell line RINm5F and rat pancreatic islets, the RLC was basally phosphorylated on the myosin light chain kinase sites (Ser19/Thr18). Phosphorylation at these sites was not consistently increased by either metabolic stimuli (glyceraldehyde/glucose) or the depolarizing agent KCl. The RLC sites phosphorylated by protein kinase C (PKC) (Ser1/Ser2) were unphosphorylated in the basal state, not affected by nutrients or KCl, and only slightly increased by the PKC activator phorbol 12-myristate 13-acetate (PMA). Like the other insulin secretagogues, however, PMA did promote serine phosphorylation of the myosin heavy chain (MHC) in RINm5F

Research in the Egelhoff lab focuses on dissecting the cellular machinery that drives cell migration and invasion, with emphasis on understanding the roles and regulation of myosin II. Studies in the lab are currently focused on understanding myosin II functions and regulation during cancer progression and invasive migration by cancer cells. We recently discovered novel and dramatic regulation of myosin II expression and activation when mammary gland epithelial cells respond to the cytokine TGFß - a stimulus that promotes epithelial-to-mesenchymal-transition (EMT). These studies have revealed a critical role for specific myosin II isoforms in invasive migration in breast epithelial cells and in breast cancer cells, a role that we are currently trying to understand at the cellular and biophysical levels. We use an array of biochemical, cellular, and live cell imaging approaches in our work, with emphasis on dynamic reporter tools such as GFP fusions and fluorescent recovery after photobleaching ...

Biochemical analysis in vertebrates suggests that RMLC phosphorylation on serine 19 (S19) triggers myosin II motor activity, and that phosphorylation on threonine 18 (T18) together with S19 further increases its activity (Ikebe et al., 1986). Likewise, phosphorylation of Drosophila RMLC at the position equivalent to S19 is sufficient to induce myosin II activity (Jordan and Karess, 1997; Vereshchagina et al., 2004). Consistent with these data, we show that a phosphomimetic variant of MLC-4/RMLC (MLC-4DD) fully rescues the elongation defect of mlc-4 mutants, whereas an unphosphorylatable form (MLC-4AA) fails to rescue the defect. Since the MLC-4T17A variant, which can still be phosphorylated on the adjacent serine (S18), rescued more significantly than the MLC-4S18A variant, we conclude that in C. elegans too, the serine plays a more crucial role in MLC-4/RMLC activation.. The kinase(s) that mediate RMLC phosphorylation during epithelial morphogenesis has not been systematically investigated ...

During amoeboid locomotion, which many metazoan cells use to migrate through tissues, actin polymerization is thought to generate filopodia and lamellipodia at the leading edge, which move the cell forward. However, this model does not fully explain amoeboid movement - for example, why do some cells glide and others move jerkily? On p. 3833, Kunito Yoshida and Thierry Soldati propose that the focal production of blebs - transparent, spherical cell-surface protrusions - is also important for amoeboid locomotion. Blebs `blow out from the cell surface when myosin-II-driven contraction of the back of the cell increases the cytoplasmic fluid pressure. By visualizing the dynamics of F-actin, the authors show that migrating Dictyostelium cells continuously produce these blebs at the leading edge. Pseudopodia extension, cell-body retraction and the speed of cell locomotion are all reduced in myosin-II-null cells, cells treated with the myosin II inhibitor blebbistatin, and cells at high osmolarity - ...

The Drosophila body axes are established in the oocyte during oogenesis. Oocyte polarization is initiated by Gurken, which signals from the germline through the epidermal growth factor receptor (Egfr) to the posterior follicle cells (PFCs). In response the PFCs generate an unidentified polarizing signal that regulates oocyte polarity. We have identified a loss-of-function mutation of flapwing, which encodes the catalytic subunit of Protein Phosphatase 1β (PP1β) that disrupts oocyte polarization. We show that PP1β, by regulating myosin activity, controls the generation of the polarizing signal. Excessive myosin activity in the PFCs causes oocyte mispolarization and defective Notch signaling and endocytosis in the PFCs. The integrated activation of JAK/STAT and Egfr signaling results in the sensitivity of PFCs to defective Notch. Interestingly, our results also demonstrate a role of PP1β in generating the polarizing signal independently of Notch, indicating a direct involvement of somatic ...

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In this study, we demonstrate that Shh protein induces macropinocytosis in the axons, through activation of a noncanonical signaling pathway. Rho GTPase and nonmuscle myosin II activities were rapidly increased during the treatment of Shh. Macropinocytosis induced by Shh was observed to occur rapidly in the growth cones and was further characterized as independent of clathrin and PI3K but dependent on dynamin and myosin II activities. Inhibitors of macropinocytosis abolished growth cone collapse and repulsive axon turning induced by high Shh, and pharmacologically increased macropinocytosis correlated with growth cone collapse and axon repulsive turning. These results support that macropinocytosis-mediated membrane trafficking is essential for chemorepulsive axon guidance.. Although the correlation of macropinocytosis and axon growth cone collapse has been reported by previous studies (Fournier et al., 2000; Jurney et al., 2002), our study provides the first functional evidence that ...

The Cabernard lab and collaborators recently published in Developmental Cell and found that cell and tissue morphogenesis depends on the correct regulation of non-muscle Myosin II, but how this motor protein is spatiotemporally controlled is incompletely understood. They show that in asymmetrically dividing Drosophila neural stem cells, cell intrinsic polarity cues provide spatial and temporal information to regulate biased Myosin activity. Using live cell imaging and a genetically encoded Myosin activity sensor, they found that Drosophila Rho kinase (Rok) enriches for activated Myosin on the neuroblast cortex prior to nuclear envelope breakdown (NEB). After NEB, the conserved polarity protein Partner of Inscuteable (Pins) sequentially enriches Rok and Protein Kinase N (Pkn) on the apical neuroblast cortex. Their data suggest that apical Rok first increases phospho-Myosin, followed by Pkn-mediated Myosin downregulation, possibly through Rok inhibition. They propose that polarity-induced ...

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For example, among those chemotherapeutic agents that work by inhibiting mitosis, many are effective anticancer agents because they interfere with microtubule function responsible for cell proliferation ( 17). However, microtubule disruption affects many important cellular processes including intracellular transport, maintenance of cell shape, cell signaling, and cell motility; therefore, the current antimitotic drugs suffer from serious side effects, including peripheral neuropathy ( 18). Work at the ICG has uncovered several new small molecules that disrupt additional key steps in cell division. The small molecules monastrol, blebbistatin, and binucleine 2 were discovered using cell-based phenotypic high-content screens ( 19), screens for non-muscle myosin II ATPase activity ( 20) and parallel RNAi and chemical genetic screens ( 21), respectively. As novel tools and lead compounds for biological study and pharmaceutical development, these compounds have the potential to evolve into ...

Myosins are motor proteins that convert chemical energy into mechanical work, generating force and movement. Myosin II generates forces that are essential to drive cell movements and cell shape changes that generate tissue structure. While researchers know that mutations in the genes that encode nonmuscle myosin II lead to diseases, including severe congenital defects as well as blood platelet dysfunction, nephritis, and deafness in adults, they do not fully understand the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology.

2007). is sufficient to mislocalize DRP1. Previous studies have demonstrated reduced organelle motility following excessive F-actin stabilization (Chada and Hollenbeck, 2004; Semenova et al., 2008). Also supporting our model, myosin II-mediated linkage of mitochondria with actin has recently been reported (Reyes et al., 2011), and mitochondria have been shown to undergo myosin-mediated transport on actin filaments in mammalian cells (Quintero Veliparib research buy et al., 2009). Alternatively, excessive F-actin within the cell might sequester DRP1 away from mitochondria. However, we do not favor this second model because destabilization of actin, like excessive stabilization, causes DRP1 mislocalization and mitochondrial elongation, as documented here (Figure 5) and previously reported by De Vos et al. (2005). Importantly, the mechanisms we outline here appear to be general ones. We observe altered mitochondrial dynamics following expression of not only FTDP-17-associated forms of tau (Figure ...

In Saccharomyces cerevisiae, it is well established that Hof1, Cyk3, and Inn1 contribute to septum formation and cytokinesis. Because hof1∆ and cyk3∆ single mutants have relatively mild defects but hof1∆ cyk3∆ double mutants are nearly dead, it has been hypothesized that these proteins contribute to parallel pathways. However, there is also evidence that they interact physically. In this study, we examined this interaction and its functional significance in detail. Our data indicate that the interaction (i) is mediated by a direct binding of the Hof1 SH3 domain to a proline-rich motif in Cyk3; (ii) occurs specifically at the time of cytokinesis but is independent of the (hyper)phosphorylation of both proteins that occurs at about the same time; (iii) is dispensable for the normal localization of both proteins; (iv) is essential for normal primary-septum formation and a normal rate of cleavage-furrow ingression; and (v) becomes critical for growth when either Inn1 or the type II myosin ...

This gene encodes a protein that is similar to a tumor suppressor in Drosophila. The protein is part of a cytoskeletal network and is associated with nonmuscle myosin II heavy chain and a kinase that specifically phosphorylates this protein at serine residues. The gene is located within the Smith-Magenis syndrome region on chromosome 17. [provided by RefSeq, Jul 2008 ...

Marked sarcomere disorganization is a well-documented characteristic of cardiomyocytes in the failing human myocardium. in cardiogenesis. Introduction Despite recent advances in pharmacologic and surgical therapies, chronic heart failure (CHF) is still a leading cause of death worldwide (1). Currently, heart transplant is thought to be the most effective therapy for end-stage CHF. However, this approach obviously cannot be used for all of the numerous affected patients and is not suitable for patients with a mild disease state. Therefore, there is increasing demand for new therapeutic targets for CHF. Cardiomyocytes, the most basic cellular unit of the myocardium, express several sarcomeric proteins, including myosin and actin; abnormalities in these sarcomeric proteins are major causes of idiopathic cardiomyopathies and lead to CHF (2C4). Type II myosin is the major constituent of sarcomeres. In the neck region of this protein, there are binding sites for a pair of myosin light chains, which ...

The subfamily of myosin proteins that are commonly found in muscle fibers. Myosin II is also involved a diverse array of cellular functions including cell division, transport within the GOLGI APPARATUS, and maintaining MICROVILLI structure ...

Background: Obesity is becoming an increasing problem in obstetric practice; it has led to an increase in the risk of caesarean delivery, prolonged pregnancy and dysfunctional labour. It has been postulated that many of ...

COA of Blebbistatin contains the actual results obtained from testing performed as part of quality control. View our Blebbistatin specific physical and chemical properties, and analytical data.

Myosin is a highly conserved, ubiquitous protein found in all eukaryotic cells, where it provides the motor function for diverse movements such as cytokinesis, phagocytosis, and muscle contraction. All myosins contain an amino-terminal motor/head domain and a carboxy-terminal tail domain. The class II myosins, consist

Amino Acid Sequence: MSSKKAKTKT TKKRPQRATS NVFAMFDQSQ IQEFKEAFNM IDQNRDGFID KEDLHDMLAS LGKNPTDAYL DAMMNEAPGP INFTMFLTMF GEKLNGTDPE DVIRNAFACF DEEATGTIQE DYLRELLTTM GDRFTDEEVD ELYREAPIDK KGNFNYIEFT RILKHGAKDK DD ...

Kumar, Abhishek and Maitra, Ananyo and Sumit, Madhuresh and Ramaswamy, Sriram and Shivashankar, G. V. (2014) Actomyosin contractility rotates the cell nucleus. In: SCIENTIFIC REPORTS, 4 . ...

Abstract: Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an ~40-fold increase in the initial ...

Abstract: Myosin heavy chain kinase (MHCK) A phosphorylates mapped sites at the C-terminal tail of Dictyostelium myosin II heavy chain, driving disassembly of myosin filaments both in vitro and in vivo. MHCK A is organized into three functional domains that include an N-terminal coiled-coil region, a central kinase catalytic domain unrelated to conventional protein kinases, and a WD repeat domain at the C terminus. MHCK B is a homologue of MHCK A that possesses structurally related catalytic and WD repeat domains. In the current study, we explored the role of the WD repeat domains in defining the activities of both MHCK A and MHCK B using recombinant bacterially expressed truncations of these kinases either with or without their WD repeat domains. We demonstrate that substrate targeting is a conserved function of the WD repeat domains of both MHCK A and MHCK B and that this targeting is specific forDictyostelium myosin II filaments. We also show that the mechanism of targeting involves direct ...

Both smooth muscle and nonmuscle myosin II activity is regulated by the phosphorylation state of the myosin regulatory light chain (MLC, MRLC, MLC20, Myl9). Phosphorylation of MLC at Thr-18 and Ser-19 activates myosin II motor activity and increases myosin filament stability. This activation has important roles in vari

TY - JOUR. T1 - The role of myosin II in glioma invasion of the brain. AU - Beadle, Christopher. AU - Assanah, Marcela C.. AU - Monzo, Pascale. AU - Vallee, Richard. AU - Rosenfeld, Steven S.. AU - Canoll, Peter. PY - 2008/8. Y1 - 2008/8. N2 - The ability of gliomas to invade the brain limits the efficacy of standard therapies. In this study, we have examined glioma migration in living brain tissue by using two novel in vivo model systems. Within the brain, glioma cells migrate like nontransformed, neural progenitor cells - extending a prominent leading cytoplasmic process followed by a burst of forward movement by the cell body that requires myosin II. In contrast, on a two-dimensional surface, glioma cells migrate more like fibroblasts, and they do not require myosin II to move. To explain this phenomenon, we studied glioma migration through a series of synthetic membranes with defined pore sizes. Our results demonstrate that the A and B isoforms of myosin II are specifically required when a ...

TY - JOUR. T1 - Myosin II contributes to cell-scale actin network treadmilling through network disassembly. AU - Wilson, Cyrus A.. AU - Tsuchida, Mark A.. AU - Allen, Greg M.. AU - Barnhart, Erin L.. AU - Applegate, Kathryn T.. AU - Yam, Patricia T.. AU - Ji, Lin. AU - Keren, Kinneret. AU - Danuser, Gaudenz. AU - Theriot, Julie A.. PY - 2010/5/20. Y1 - 2010/5/20. N2 - Crawling locomotion of eukaryotic cells is achieved by a process dependent on the actin cytoskeleton: protrusion of the leading edge requires assembly of a network of actin filaments, which must be disassembled at the cell rear for sustained motility. Although ADF/cofilin proteins have been shown to contribute to actin disassembly, it is not clear how activity of these locally acting proteins could be coordinated over the distance scale of the whole cell. Here we show that non-muscle myosin II has a direct role in actin network disassembly in crawling cells. In fish keratocytes undergoing motility, myosin II is concentrated in ...

Anyway, what the oxidized product does is to stop the process of cytokinesis in place. So you can freeze frame the process of cytokinesis at any timepoint where you add a drop of the compound. If you wash it out, cytokinesis picks up where it left off.. The mechanism of action turns out to be that furrowstatin binds a protein called non-muscle myosin II. This showed that this motor protein is involved in cytokinesis, but is not involved in sister chromatid separation, since that process could continue in the presence of furrowstatin.. Once this was known, people started staining for non-muscle myosin II, and found that it co-localized with actin during cytokinesis. Then they stained for other things and found that anillin co-localized with actin as well, suggesting it might also be involved.. A phenotypic screen revealed a second tool compound, which caused a phenotype never before seen in nature: it made segregating chromosomes and microtubules explode into an astral shape, with the centromeres ...

TY - JOUR. T1 - Myosin II is involved in the production of constitutive transport vesicles from the TGN. AU - Müsch, Anne. AU - Cohen, David. AU - Rodriguez-Boulan, Enrique. PY - 1997/7/28. Y1 - 1997/7/28. N2 - The participation of nonmuscle myosins in the transport of organelles and vesicular carriers along actin filaments has been documented. In contrast, there is no evidence for the involvement of myosins in the production of vesicles involved in membrane traffic. Here we show that the putative TGN coat protein p200 (Narula, N., I. McMorrow, G. Plopper, J. Doherty, K.S. Matlin, B. Burke, and J.L. Stow. 1992. J. Cell Biol. 114: 1113- 1124) is myosin II. The recruitment of myosin II to Golgi membranes is dependent on actin and is regulated by G proteins. Using an assay that studies the release of transport vesicles from the TGN in vitro we provide functional evidence that p200/myosin is involved in the assembly of basolateral transport vesicles carrying vesicular stomatitis virus G protein ...

DISCUSSION. Myosin RLC phosphorylation at Ser19 (P-RLC) is sufficient to fully activate SMMII and NMII (reviewed in [27]), however frequently observed RLC di-phosphorylation at Thr18/Ser19 (PP-RLC) (reviewed in [28]) may be responsible for prolonged myosin activation because of significantly slower RLC dephosphorylation [29]. Based on these findings, mono-phosphorylation at Ser19 and/or di-phosphorylation at Thr18/Ser19 are widely used to biochemically characterize myosin II motor activity and its involvement in (patho)physiological responses.. A variety of experimental approaches has been developed for analysis of P-RLC and PP-RLC. One of these approaches is electrophoretic separation of non-phosphorylated RLC, P-RLC, and PP-RLC followed by staining with a protein dye and densitometric analysis of the stained protein bands. RLC phospho-species can be separated by isoelectric focusing in the presence of pyrophosphate [9], urea/glycerol polyacrylamide electrophoresis [10], or recently introduced ...

Amoeboid motility requires spatiotemporal coordination of biochemical pathways regulating force generation and consists of the quasi-periodic repetition of a EPZ-6438 motility cycle driven by actin polymerization and actomyosin contraction. tensional stress and that wild-type cells develop two opposing EPZ-6438 pole forces pulling the front and back toward the center whose strength is modulated up and down periodically in each cycle. We demonstrate that nonmuscular myosin II complex (MyoII) cross-linking and motor functions have different roles in controlling the spatiotemporal distribution of traction forces the changes in cell shape and the duration of all the phases. We show that the time required to complete each phase is dramatically increased in cells with altered MyoII motor function demonstrating that it is required not only for contraction but also for protrusion. Concomitant loss of MyoII actin cross-linking leads to a force redistribution throughout the cell perimeter pulling inward ...

Serotonin (5-HT) is known to increase the rate of growth cone advance via cofilin-dependent increases in retrograde actin network flow and nonmuscle myosin II activity ...

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical ...

The Laboratory of Molecular Cardiology, led by Dr. Robert S. Adelstein, is focused on the role of non-muscle myosin II (NM II) in development and disease ...

Recombinant human MRCKβ (amino acids 1-473) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. Myotonic Dystrophy Kinase-Related cdc42-binding kinase beta (MRCKβ) belongs to the DMPK subfamily. Myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) is an effector of Cdc42, where it is involved in actin cytoskeletal reorganization and neurite outgrowth.

Contractile forces are important for cell function. Forces are transmitted by the cytoskeleton, a dynamic scaffold of protein filaments throughout the cytoplasm connected to the plasma membrane. Actin and Myosin II have been identified as the main components in this contraction. F-actin provides the structure upon which myosin performs its job, powered by ATP hydrolysis.Contraction of cross-linked actin-myosin networks is mediated by internal stresses that are actively generated by the myosin motors. Myosin assembles and generates gliding of actin filaments past one another. Though there have been many successful attempts to model the contractile actin cortex, there is still a limited understanding of the dependence of contractility and pattern formation in actin-myosin gels on microscopic parameters such as the number, activity, and processivity of the myosin motors or the local cross-linker density and actin network connectivity. Experiments have shown that contraction is accelerated by ...

It had been known from previous work that the motor protein myosin would synthesise ATP spontaneously from ADP and phosphate. However, this ATP remained irreversibly bound to the protein unless an external force was applied (3,6). They then surmised that if ATP-synthase could be rotated manually, the ATP that was generated spontaneously, from ADP and inorganic phosphate, might then be released. Itoh and colleagues took the axle and the F1 subunit, link the axle to a magnetic bead and the F1 head to a surface via histidine tags. Using an externally applied magnetic field the bead was rotated, dragging the g-subunit with it. In a medium enriched in ADP and inorganic phosphate, ATP synthesis was detected through luciferase activity. For every rotation through 120o, at a frequency of 3Hz, 5 molecules of ATP were synthesised - roughly half of the expected 9 ATP molecules observed in vitro (5). (By contrast, the reverse ATP-hydrolysis reaction drove the bead at a maximum rotation frequency of 130Hz ...

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In this review, recent progress relating to the integration of nuclear events was discussed, e.g. deciphering how RNAP II functions in the various RNA processing reactions needed to synthesize …

Function VisibleCells(Rng As Range) As Variant VisibleCells This function returns an array equal in dimension to the input parameter Rng containing 1s and 0s indicating whether a cells within Rng is visible. Note that we use 1 to indicate True rather than VBAs True value (which equals -1). If Rng has more than one area (discontiguous ranges), the function returns a #REF error. Dim R As Range Dim Arr() As Integer Dim RNdx As Long Dim CNdx As Long Ensure a valid range. If Rng.Areas.Count > 1 Then VisibleCells = CVErr(xlErrRef) Exit Function End If Size the return array to equal the Rng parameter. ReDim Arr(1 To Rng.Rows.Count, 1 To Rng.Columns.Count) For RNdx = 1 To Rng.Rows.Count For CNdx = 1 ...

4DBQ: Mutations in myosin VI that cause a loss of coordination between heads provide insights into the structural changes underlying force generation and the importance of gating

4DBP: Mutations in myosin VI that cause a loss of coordination between heads provide insights into the structural changes underlying force generation and the importance of gating