Peptide Probes for Plasmodium falciparum MyoA Tail Interacting Protein (MTIP): Exploring the Druggability of the Malaria Parasite Motor Complex.

Malaria remains an endemic tropical disease, and the emergence of Plasmodium falciparum parasites resistant to current front-line medicines means that new therapeutic targets are required. The Plasmodium glideosome is a multiprotein complex thought to be essential for efficient host red blood cell invasion. At its core is a myosin motor, Myosin A (MyoA), which provides most of the force required for parasite invasion. Here, we report the design and development of improved peptide-based probes for the anchor point of MyoA, the P. falciparum MyoA tail interacting protein (PfMTIP). These probes combine low nanomolar binding affinity with significantly enhanced cell penetration and demonstrable competitive target engagement with native PfMTIP through a combination of Western blot and chemical proteomics. These results provide new insights into the potential druggability of the MTIP/MyoA interaction and a basis for the future design of inhibitors.

Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion.

Filopodia, dynamic membrane protrusions driven by polymerization of an actin filament core, can adhere to the extracellular matrix and experience both external and cell-generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we study filopodia induced by overexpression of myosin X, typical for cancer cells. The lifetime of such filopodia positively correlates with the presence of myosin IIA filaments at the filopodia bases. Application of pulling forces to the filopodia tips through attached fibronectin-coated laser-trapped beads results in sustained growth of the filopodia. Pharmacological inhibition or knockdown of myosin IIA abolishes the filopodia adhesion to the beads. Formin inhibitor SMIFH2, which causes detachment of actin filaments from formin molecules, produces similar effect. Thus, centripetal force generated by myosin IIA filaments at the base of filopodium and transmitted to the tip through actin core in a formin-dependent fashion is required for filopodia adhesion.

The role of nonmuscle myosin II in polydrug memories and memory reconsolidation.

Using pharmacologic and genetic approaches targeting actin or the actin-driving molecular motor, nonmuscle myosin II (NMII), we previously discovered an immediate, retrieval-independent, and long-lasting disruption of methamphetamine- (METH-) and amphetamine-associated memories. A single intrabasolateral amygdala complex infusion or systemic administration of the NMII inhibitor Blebbistatin (Blebb) is sufficient to produce this disruption, which is selective, having no retrieval-independent effect on memories for fear, food reward, cocaine, or morphine. However, it was unclear if Blebb treatment would disrupt memories of other stimulants and amphetamine class drugs, such as nicotine (NIC) or mephedrone (MEPH; bath salts). Moreover, many individuals abuse multiple drugs, but it was unknown if Blebb could disrupt polydrug memories, or if the inclusion of another substance would render Blebb no longer able to disrupt METH-associated memories. Therefore, the present study had two primary goals: (1) to determine the ability of Blebb to disrupt NIC- or MEPH-associated memories, and (2) to determine the ability of METH to modify other unconditioned stimulus (US) associations' susceptibility to Blebb. To this end, using the conditional place preference model, mice were conditioned to NIC and MEPH alone or METH in combination with NIC, morphine, or foot shock. We report that, unlike METH, there was no retrieval-independent effect of Blebb on NIC- or MEPH-associated memories. However, similar to cocaine, reconsolidation of the memory for both drugs was disrupted. Further, when combined with METH administration, NIC- and morphine-, but not fear-, associated memories were rendered susceptible to disruption by Blebb. Given the high rate of polydrug use and the resurgence of METH use, these results have important implications for the treatment of substance use disorder.

Phosphorylation of Nonmuscle myosin II-A regulatory light chain resists Sendai virus fusion with host cells.

Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion.

NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3ß-NF-κB signalling pathways in the endothelium.

Non-muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor-α (TNF-α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose-dependent manner. Blebbistatin enhanced Akt and GSK3ß phosphorylation and inhibited NF-κB p65 nuclear translocation and IκBα degradation. These observations were similar to the effect of CHIR99021, a GSK3ß inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3ß and suppressed NF-κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF-α-stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3ß by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model.Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3ß-NF-κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders.

Pigment granule translocation in red ovarian chromatophores from the palaemonid shrimp Macrobrachium olfersi (Weigmann, 1836): functional roles for the cytoskeleton and its molecular motors.

The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca²âº/cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro. Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells.

Golgi fragmentation induced by heat shock or inhibition of heat shock proteins is mediated by non-muscle myosin IIA via its interaction with glycosyltransferases.

The Golgi apparatus is a highly dynamic organelle which frequently undergoes morphological changes in certain normal physiological processes or in response to stress. The mechanisms are largely not known. We have found that heat shock of Panc1 cells expressing core 2 N-acetylglucosaminyltransferase-M (Panc1-C2GnT-M) induces Golgi disorganization by increasing non-muscle myosin IIA (NMIIA)-C2GnT-M complexes and polyubiquitination and proteasomal degradation of C2GnT-M. These effects are prevented by inhibition or knockdown of NMIIA. Also, the speed of Golgi fragmentation induced by heat shock is found to be positively correlated with the levels of C2GnT-M in the Golgi. The results are reproduced in LNCaP cells expressing high levels of two endogenous glycosyltransferases-core 2 N-acetylglucosaminyltransferase-L:1 and ß-galactoside:α2-3 sialyltransferase 1. Further, during recovery after heat shock, Golgi reassembly as monitored by a Golgi matrix protein giantin precedes the return of C2GnT-M to the Golgi. The results are consistent with the roles of giantin as a building block of the Golgi architecture and a docking site for transport vesicles carrying glycosyltransferases. In addition, inhibition/depletion of HSP70 or HSP90 in Panc1-C2GnT-M cells also causes an increase of NMIIA-C2GnT-M complexes and NMIIA-mediated Golgi fragmentation but results in accumulation or degradation of C2GnT-M, respectively. These results can be explained by the known functions of these two HSP: participation of HSP90 in protein folding and HSP70 in protein folding and degradation. We conclude that NMIIA is the master regulator of Golgi fragmentation induced by heat shock or inhibition/depletion of HSP70/90.

MYBPH inhibits NM IIA assembly via direct interaction with NMHC IIA and reduces cell motility.

Actomyosin filament assembly is a critical step in tumor cell migration. We previously found that myosin binding protein H (MYBPH) is directly transactivated by the TTF-1 lineage-survival oncogene in lung adenocarcinomas and inhibits phosphorylation of the myosin regulatory light chain (RLC) of non-muscle myosin IIA (NM IIA) via direct interaction with Rho kinase 1 (ROCK1). Here, we report that MYBPH also directly interacts with an additional molecule, non-muscle myosin heavy chain IIA (NMHC IIA), which was found to occur between MYBPH and the rod portion of NMHC IIA. MYBPH inhibited NMHC IIA assembly and reduced cell motility. Conversely, siMYBPH-induced increased motility was partially, yet significantly, suppressed by blebbistatin, a non-muscle myosin II inhibitor, while more profound effects were attained by combined treatment with siROCK1 and blebbistatin. Electron microscopy observations showed well-ordered paracrystals of NMHC IIA reflecting an assembled state, which were significantly less frequently observed in the presence of MYBPH. Furthermore, an in vitro sedimentation assay showed that a greater amount of NMHC IIA was in an unassembled state in the presence of MYBPH. Interestingly, treatment with a ROCK inhibitor that impairs transition of NM IIA from an assembly-incompetent to assembly-competent state reduced the interaction between MYBPH and NMHC IIA, suggesting that MYBPH has higher affinity to assembly-competent NM IIA. These results suggest that MYBPH inhibits RLC and NMHC IIA, independent components of NM IIA, and negatively regulates actomyosin organization at 2 distinct steps, resulting in firm inhibition of NM IIA assembly.

Blood clots are rapidly assembled hemodynamic sensors: flow arrest triggers intraluminal thrombus contraction.

OBJECTIVE: Blood clots form under flow during intravascular thrombosis or vessel leakage. Prevailing hemodynamics influence thrombus structure and may regulate contraction processes. A microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) was used to measure thrombus permeability (κ) and contraction at controlled transthrombus pressure drops. METHODS AND RESULTS: The collagen (κ(collagen)=1.98 × 10(-11) cm(2)) supported formation of a 20-µm thick platelet layer, which unexpectedly underwent massive platelet retraction on flow arrest. This contraction resulted in a 5.34-fold increase in permeability because of collagen restructuring. Without stopping flow, platelet deposits (no fibrin) had a permeability of κ(platelet)=5.45 × 10(-14) cm(2) and platelet-fibrin thrombi had κ(thrombus)=2.71 × 10(-14) cm(2) for ΔP=20.7 to 23.4 mm Hg, the first ever measurements for clots formed under arterial flow (1130 s(-1) wall shear rate). Platelet sensing of flow cessation triggered a 4.6- to 6.5-fold (n=3, P<0.05) increase in contraction rate, which was also observed in a rigid, impermeable parallel-plate microfluidic device. This triggered contraction was blocked by the myosin IIA inhibitor blebbistatin and by inhibitors of thromboxane A2 (TXA(2)) and ADP signaling. In addition, flow arrest triggered platelet intracellular calcium mobilization, which was blocked by TXA(2)/ADP inhibitors. As clots become occlusive or blood pools following vessel leakage, the flow diminishes, consequently allowing full platelet retraction. CONCLUSIONS: Flow dilution of ADP and thromboxane regulates platelet contractility with prevailing hemodynamics, a newly defined flow-sensing mechanism to regulate clot function.

Hyaluronic acid matrices show matrix stiffness in 2D and 3D dictates cytoskeletal order and myosin-II phosphorylation within stem cells.

Physical features of microenvironments such as matrix elasticity E can clearly influence cell morphology and cell phenotype, but many differences between model matrices raise questions as to whether a standard biological scale for E exists, especially in 3D as well as in 2D. An E-series of two distinct types of hydrogels are ligand-functionalized here with non-fibrous collagen and used to elucidate wide-ranging cell and cytoskeletal responses to E in both 2D and 3D matrix geometries. Cross-linked hyaluronic acid (HA) based matrices as well as standard polyacrylamide (PA) hydrogels show that, within hours of initial plating, the adhesion, asymmetric shape, and cytoskeletal order within mesenchymal stem cells generally depend on E nonmonotonically over a broad range of physiologically relevant E. In particular, with overlays of a second matrix the stiffer of the upper or lower matrix dominates key cell responses to 3D: the cell invariably takes an elongated shape that couples to E in driving cytoplasmic stress fiber assembly. In contrast, embedding cells in homogeneous HA matrices constrains cells to spherically symmetric shapes in which E drives the assembly of a predominantly cortical cytoskeleton. Non-muscle myosin II generates the forces required for key cell responses and is a target of a phospho-Tyrosine signaling pathway that likely regulates contractile assemblies and also depends nonmonotonically on E. The results can be understood in part from a theory for stress fiber polarization that couples to matrix elasticity as well as cell shape and accurately predicts cytoskeletal order in 2D and 3D, regardless of polymer system.

Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens.

Anaplastic gliomas, the most common and malignant of primary brain tumors, frequently contain activating mutations and amplifications in promigratory signal transduction pathways. However, targeting these pathways with individual signal transduction inhibitors does not appreciably reduce tumor invasion, because these pathways are redundant; blockade of any one pathway can be overcome by stimulation of another. This implies that a more effective approach would be to target a component at which these pathways converge. In this study, we have investigated whether the molecular motor myosin II represents such a target by examining glioma invasion in a series of increasingly complex models that are sensitive to platelet-derived growth factor, epidermal growth factor, or both. Our results lead to two conclusions. First, malignant glioma cells are stimulated to invade brain through the activation of multiple signaling cascades not accounted for in simple in vitro assays. Second, even though there is a high degree of redundancy in promigratory signaling cascades in gliomas, blocking tumor invasion by directly targeting myosin II remains effective. Our results thus support our hypothesis that myosin II represents a point of convergence for signal transduction pathways that drive glioma invasion and that its inhibition cannot be overcome by other motility mechanisms.

Enucleation of human erythroblasts involves non-muscle myosin IIB.

Mammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34(+) cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.

Cysteine 81 is critical for the interaction of S100A4 and myosin-IIA.

Overexpression of S100A4, a member of the S100 family of Ca(2+)-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca(2+)-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes.

Cell division, membrane rigidity, and strong adhesion to a rigid matrix are all promoted by myosin-II, and so multinucleated cells with distended membranes--typical of megakaryocytes (MKs)--seem predictable for low myosin activity in cells on soft matrices. Paradoxically, myosin mutations lead to defects in MKs and platelets. Here, reversible inhibition of myosin-II is sustained over several cell cycles to produce 3- to 10-fold increases in polyploid MK and a number of other cell types. Even brief inhibition generates highly distensible, proplatelet-like projections that fragment readily under shear, as seen in platelet generation from MKs in vivo. The effects are maximized with collagenous matrices that are soft and 2D, like the perivascular niches in marrow rather than 3D or rigid, like bone. Although multinucleation of other primary hematopoietic lineages helps to generalize a failure-to-fission mechanism, lineage-specific signaling with increased polyploidy proves possible and novel with phospho-regulation of myosin-II heavy chain. Label-free mass spectrometry quantitation of the MK proteome uses a unique proportional peak fingerprint (ProPF) analysis to also show upregulation of the cytoskeletal and adhesion machinery critical to platelet function. Myosin-inhibited MKs generate more platelets in vitro and also in vivo from the marrows of xenografted mice, while agonist stimulation activates platelet spreading and integrin αIIbß3. Myosin-II thus seems a central, matrix-regulated node for MK-poiesis and platelet generation.

An experimental model for studying the biomechanics of embryonic tendon: Evidence that the development of mechanical properties depends on the actinomyosin machinery.

Tendons attach muscles to bone and thereby transmit tensile forces during joint movement. However, a detailed understanding of the mechanisms that establish the mechanical properties of tendon has remained elusive because of the practical difficulties of studying tissue mechanics in vivo. Here we have performed a study of tendon-like constructs made by culturing embryonic tendon cells in fixed-length fibrin gels. The constructs display mechanical properties (toe-linear-fail stress-strain curve, stiffness, ultimate tensile strength, and failure strain) as well as collagen fibril volume fraction and extracellular matrix (ECM)/cell ratio that are statistically similar to those of embryonic chick metatarsal tendons. The development of mechanical properties during time in culture was abolished when the constructs were treated separately with Triton X-100 (to solubilise membranes), cytochalasin (to disassemble the actin cytoskeleton) and blebbistatin (a small molecule inhibitor of non-muscle myosin II). Importantly, these treatments had no effect on the mechanical properties of the constructs that existed prior to treatment. Live-cell imaging and (14)C-proline metabolic labeling showed that blebbistatin inhibited the contraction of the constructs without affecting cell viability, procollagen synthesis, or conversion of procollagen to collagen. In conclusion, the mechanical properties per se of the tendon constructs are attributable to the ECM generated by the cells but the improvement of mechanical properties during time in culture was dependent on non-muscle myosin II-derived forces.

Inhibiting nonmuscle myosin II impedes inflammatory infiltration and ameliorates progressive renal disease.

The motor protein nonmuscle myosin II (NMII) through its interaction with the actin cytoskeleton constitutes the machinery of cell crawling and has an important role in driving locomotion and infiltration of immune competent cells during inflammatory response and immune reaction. Blebbistatin is a highly selective inhibitor of NMII adenosine triphosphatase. This study examined the effect of NMII inhibition by blebbistatin on inflammation. In vitro, blebbistatin markedly induced actinomyosin complex disassembly in various cultured immunocytes, and functionally impaired their motile activity and invasive capacity as assessed by the Boyden chamber motility assay and the matrigel invasion assay. In vivo, in a rat model of acute inflammation induced by tumor necrosis factor, blebbistatin obliterated renal sequestration of circulating fluorescence-labeled macrophages in a dose-dependent fashion. Moreover, in rats with progressive obstructive nephropathy, blebbistatin treatment exhibited a remarkable renoprotective effect, as evidenced by normalized kidney weight, improved gross morphology, and diminished histologic injury in the tubulointerstitium. This beneficial effect was associated with significant amelioration of renal inflammation, consistent with a primary anti-inflammatory action by blebbistatin. In addition, in rats with established obstructive nephropathy, blebbistatin pretreated macrophages showed obliterated recruitment into the inflamed renal parenchyma, denoting that blebbistatin directly impedes inflammatory infiltration by immunocytes. Collectively, our findings suggest that inhibition of NMII has a potent and direct anti-inflammatory effect on the basis of impairment of the actinomyosin powered locomotive machinery, which is essential for migration and infiltration of immune competent cells.

Mechanosensing in actin stress fibers revealed by a close correlation between force and protein localization.

The mechanics of the actin cytoskeleton have a central role in the regulation of cells and tissues, but the details of how molecular sensors recognize deformations and forces are elusive. By performing cytoskeleton laser nanosurgery in cultured epithelial cells and fibroblasts, we show that the retraction of stress fibers (SFs) is restricted to the proximity of the cut and that new adhesions form at the retracting end. This suggests that SFs are attached to the substrate. A new computational model for SFs confirms this hypothesis and predicts the distribution and propagation of contractile forces along the SF. We then analyzed the dynamics of zyxin, a focal adhesion protein present in SFs. Fluorescent redistribution after laser nanosurgery and drug treatment shows a high correlation between the experimentally measured localization of zyxin and the computed localization of forces along SFs. Correlative electron microscopy reveals that zyxin is recruited very fast to intermediate substrate anchor points that are highly tensed upon SF release. A similar acute localization response is found if SFs are mechanically perturbed with the cantilever of an atomic force microscope. If actin bundles are cut by nanosurgery in living Drosophila egg chambers, we also find that zyxin redistribution dynamics correlate to force propagation and that zyxin relocates at tensed SF anchor points, demonstrating that these processes also occur in living organisms. In summary, our quantitative analysis shows that force and protein localization are closely correlated in stress fibers, suggesting a very direct force-sensing mechanism along actin bundles.

Mertk drives myosin II redistribution during retinal pigment epithelial phagocytosis.

PURPOSE: Mertk is a key phagocytic receptor in the immune, male reproductive, and visual systems. In the retinal pigment epithelium, Mertk is required for the daily ingestion of photoreceptor outer segment (OS) tips. Loss of Mertk function causes retinal degeneration in rats, mice, and humans; however, little is known about the mechanism by which Mertk regulates the ingestion phase of retinal pigment epithelial (RPE) phagocytosis. To address this, the authors sought proteins that associated with Mertk during OS phagocytosis. METHODS: Lysates of RPE-J cells challenged with OS for various times were immunoprecipitated with Mertk antibody. Potential interacting proteins were identified by mass spectrometry and characterized with confocal microscopy, pharmacologic inhibition, and siRNA knockdown coupled with an in vitro phagocytic assay in primary RPE cells. RESULTS: Myh9, the non-muscle myosin II-A heavy chain, was enriched in immunoprecipitates from OS-treated samples. Myosin II-A and II-B isoforms exhibited a striking redistribution in wild-type rat primary RPE cells challenged with OS, moving from the cell periphery to colocalize with ingested OS over time. In contrast, myosin II-A redistribution in response to OS was blunted in primary RPE cells from RCS rats, which lack functional Mertk. Wild-type rat primary RPE cells treated with the myosin II-specific inhibitor blebbistatin or myosin II siRNAs exhibited a significant phagocytic defect. CONCLUSIONS: Mertk mobilizes myosin II from the RPE cell periphery to sites of OS engulfment, where myosin II function is essential for the normal phagocytic ingestion of OS.

Assessment of myosin II, Va, VI and VIIa loss of function on endocytosis and endocytic vesicle motility in bone marrow-derived dendritic cells.

An essential feature of dendritic cell immune surveillance is endocytic sampling of the environment for non-self antigens primarily via macropinocytosis and phagocytosis. The role of several members of the myosin family of actin based molecular motors in dendritic cell endocytosis and endocytic vesicle movement was assessed through analysis of dendritic cells derived from mice with functionally null myosin mutations. These include the dilute (myosin Va), Snell's waltzer (myosin VI) and shaker-1 (myosin VIIa) mouse lines. Non muscle myosin II function was assessed by treatment with the inhibitor, blebbistatin. Flow cytometric analysis of dextran uptake by dendritic cells revealed that macropinocytosis was enhanced in Snell's waltzer dendritic cells while shaker-1 and blebbistatin-treated cells were comparable to controls. Comparison of fluid phase uptake using pH insensitive versus pH sensitive fluorescent dextrans revealed that in dilute cells rates of uptake were normal but endosomal acidification was accelerated. Phagocytosis, as quantified by uptake of E. coli, was normal in dilute while dendritic cells from Snell's waltzer, shaker-1 and blebbistatin treated cells exhibited decreased uptake. Microtubule mediated movements of dextran-or transferrin-tagged endocytic vesicles were significantly faster in dendritic cells lacking myosin Va. Loss of myosin II, VI or VIIa function had no significant effects on rates of endocytic vesicle movement.

Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk.

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.