Nonmuscle myosin II is a critical regulator of clathrin-mediated endocytosis.

Variable requirements for actin during clathrin-mediated endocytosis (CME) may be related to regional or cellular differences in membrane tension. To compensate, local regulation of force generation may be needed to facilitate membrane curving and vesicle budding. Force generation is assumed to occur primarily through actin polymerization. Here we examine the role of myosin II using loss of function experiments. Our results indicate that myosin II acts on cortical actin scaffolds primarily in the plane of the plasma membrane (bottom arrow) to generate changes that are critical for enhancing CME progression.

Loss of focal adhesion kinase enhances endothelial barrier function and increases focal adhesions.

OBJECTIVE: To determine the role of FAK in the regulation of endothelial barrier function. METHODS: Stable FAK knockdown HLEC were generated by lentiviral infection of FAK shRNA. Measurements of isometric tension and transendothelial electrical resistance were performed. RESULTS: A FAK knockdown human pulmonary endothelial cell line was generated by lentiviral infection with FAK shRNA and resulted in greater than 90% reduction in FAK protein with no change in Pyk2 protein. Loss of FAK altered cell morphology and actin distribution in both pre- and post-confluent endothelial cells. Large, polygonal shaped endothelial cells with randomly organized stress fibers were identified in pre-confluent cultures, while in confluent monolayers, endothelial cells were irregularly shaped with actin bundles present at cell margins. An increase in the number and size of vinculin plaques was detected in FAK-depleted cells. FAK knockdown monolayers generated a greater transendothelial electrical resistance than controls. Thrombin treatment induced similar changes in TER in both FAK knockdown and control cell lines. FAK-depleted endothelial cells developed a higher stable basal isometric tension compared to control monolayers, but the increase in tension stimulated by thrombin does not differ between the cell lines. Basal myosin II regulatory light chain phosphorylation was unaltered in FAK-depleted cells. In addition, loss of FAK enhanced VE-cadherin localization to the cell membrane without altering VE-cadherin protein levels. CONCLUSIONS: The loss of FAK in endothelial cells enhanced cell attachment and strengthened cell-cell contacts resulting in greater basal tension leading to formation of a tighter endothelial monolayer.

Myosin light chain kinase mediates transcellular intravasation of breast cancer cells through the underlying endothelial cells: a three-dimensional FRET study.

The transient and localized signaling events between invasive breast cancer cells and the underlying endothelial cells have remained poorly characterized. We report a novel approach integrating vascular engineering with three-dimensional time-lapse fluorescence resonance energy transfer (FRET) imaging to dissect how endothelial myosin light chain kinase (MLCK) is modulated during tumor intravasation. We show that tumor transendothelial migration occurs via both paracellular (i.e. through cell-cell junctions) and transcellular (i.e. through individual endothelial cells) routes. Endothelial MLCK is activated at the invasion site, leading to regional diphosphorylation of myosin-II regulatory light chain (RLC) and myosin contraction. Blocking endothelial RLC diphosphorylation blunts tumor transcellular, but not paracellular, invasion. Our results implicate an important role for endothelial myosin-II function in tumor intravasation.

Three-dimensional nonlinear microspectroscopy and imaging of soft condensed matter.

We report on the realization of a sensitive microspectroscopy and imaging approach based on a three-color femtosecond coherent anti-Stokes Raman scattering (CARS) technique with high spectral, time, and spatial resolution. Independently tunable, high-repetition rate optical parametric oscillators were used to attain a dynamic range of 5 orders of magnitude for time-domain CARS signal. The attained sensitivity permitted tracing the decay of weak and structurally complex Raman active modes in soft condensed matter. Application of this approach to imaging of the biological specimen shows a great potential in quantitative characterization of live biological media with an ability to access inter- and intra-molecular interactions.

Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance.

Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion-cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation.

Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa.

Cancer patients are known to be highly susceptible to Pseudomonas aeruginosa (Pa) infection, but it remains unknown whether alterations at the tumor cell level can contribute to infection. This study explored how cellular changes associated with tumor metastasis influence Pa infection using highly metastatic MTLn3 cells and non-metastatic MTC cells as cell culture models. MTLn3 cells were found to be more sensitive to Pa infection than MTC cells based on increased translocation of the type III secretion effector, ExoS, into MTLn3 cells. Subsequent studies found that higher levels of ExoS translocation into MTLn3 cells related to Pa entry and secretion of ExoS within MTLn3 cells, rather than conventional ExoS translocation by external Pa. ExoS includes both Rho GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT) enzyme activities, and differences in MTLn3 and MTC cell responsiveness to ExoS were found to relate to the targeting of ExoS-GAP activity to Rho GTPases. MTLn3 cell migration is mediated by RhoA activation at the leading edge, and inhibition of RhoA activity decreased ExoS translocation into MTLn3 cells to levels similar to those of MTC cells. The ability of Pa to be internalized and transfer ExoS more efficiently in association with Rho activation during tumor metastasis confirms that alterations in cell migration that occur in conjunction with tumor metastasis contribute to Pa infection in cancer patients. This study also raises the possibility that Pa might serve as a biological tool for dissecting or detecting cellular alterations associated with tumor metastasis.

Dorsal root ganglion neurons react to semaphorin 3A application through a biphasic response that requires multiple myosin II isoforms.

Growth cone responses to guidance cues provide the basis for neuronal pathfinding. Although many cues have been identified, less is known about how signals are translated into the cytoskeletal rearrangements that steer directional changes during pathfinding. Here we show that the response of dorsal root ganglion (DRG) neurons to Semaphorin 3A gradients can be divided into two steps: growth cone collapse and retraction. Collapse is inhibited by overexpression of myosin IIA or growth on high substrate-bound laminin-1. Inhibition of collapse also prevents retractions; however collapse can occur without retraction. Inhibition of myosin II activity with blebbistatin or by using neurons from myosin IIB knockouts inhibits retraction. Collapse is associated with movement of myosin IIA from the growth cone to the neurite. Myosin IIB redistributes from a broad distribution to the rear of the growth cone and neck of the connecting neurite. High substrate-bound laminin-1 prevents or reverses these changes. This suggests a model for the Sema 3A response that involves loss of growth cone myosin IIA to facilitate actin meshwork instability and collapse, followed by myosin IIB concentration at the rear of the cone and neck region where it associates with actin bundles to drive retraction.

Regulated proteolysis of nonmuscle myosin IIA stimulates osteoclast fusion.

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.

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 Ca(2+) 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.

Sphingosine 1-phosphate rapidly increases endothelial barrier function independently of VE-cadherin but requires cell spreading and Rho kinase.

Sphingosine 1-phosphate (S1P) rapidly increases endothelial barrier function and induces the assembly of the adherens junction proteins vascular endothelial (VE)-cadherin and catenins. Since VE-cadherin contributes to the stabilization of the endothelial barrier, we determined whether the rapid, barrier-enhancing activity of S1P requires VE-cadherin. Ca(2+)-dependent, homophilic VE-cadherin binding of endothelial cells, derived from human umbilical veins and grown as monolayers, was disrupted with EGTA, an antibody to the extracellular domain of VE-cadherin, or gene silencing of VE-cadherin with small interfering RNA. All three protocols caused a reduction in the immunofluorescent localization of VE-cadherin at intercellular junctions, the separation of adjacent cells, and a decrease in basal endothelial electrical resistance. In all three conditions, S1P rapidly increased endothelial electrical resistance. These findings demonstrate that S1P enhances the endothelial barrier independently of homophilic VE-cadherin binding. Junctional localization of VE-cadherin, however, was associated with the sustained activity of S1P. Imaging with phase-contrast and differential interference contrast optics revealed that S1P induced cell spreading and closure of intercellular gaps. Pretreatment with latrunculin B, an inhibitor of actin polymerization, or Y-27632, a Rho kinase inhibitor, attenuated cell spreading and the rapid increase in electrical resistance induced by S1P. We conclude that S1P rapidly closes intercellular gaps, resulting in an increased electrical resistance across endothelial cell monolayers, via cell spreading and Rho kinase and independently of VE-cadherin.

Myosin phosphatase and cofilin mediate cAMP/cAMP-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin II regulatory light chain phosphorylation.

This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 microm forskolin, 40 microm 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.

Mechanics of cell spreading: role of myosin II.

As it migrates over a substratum, a cell must exert different kinds of forces that act at various cellular locations and at specific times. These forces must therefore be coordinately regulated. The Rho-family GTPases Rac1 and Cdc42 promote actin polymerization that drives extension of the leading cell edge. Subsequently, RhoA regulates myosin-dependent contractile force, which is required for formation of adhesive contacts and stress fibers. During cell spreading, however, the activity of RhoA is reduced by a mechanism involving the tyrosine kinases c-Src and focal adhesion kinase (FAK), and the p190RhoGAP. It has been proposed that this reduction of RhoA activity facilitates edge extension by reducing myosin-dependent contractile forces that could resist this process. We have directly tested this hypothesis by correlating myosin activity with the rate of cell spreading on a substratum. The rate of spreading is inversely related to the myosin activity. Furthermore, spreading is inhibited by low concentrations of cytochalasin D, as expected for a process that depends on the growth of uncapped actin filaments. Cell indentation measurements show that a myosin-dependent viscoelastic force resists cell deformation.

Calcium-independent phospholipase A2 mediates CREB phosphorylation in double-stranded RNA-stimulated endothelial cells.

One of the products of a calcium-independent phospholipase A2 (iPLA2) attack of plasmenylcholine, lysoplasmenylcholine, has previously been shown to activate cAMP-dependent protein kinase (PKA). Because endothelial cells respond to some agonists in part by the activation of iPLA2, the present study was designed to determine whether double-stranded RNA (dsRNA), the primary activator of the antiviral response in endothelial cells, elicits cAMP response element binding protein (CREB) phosphorylation through a mechanism mediated by iPLA2. dsRNA stimulated CREB phosphorylation in bovine pulmonary artery endothelial cells that was inhibited by the iPLA2 inhibitor, bromoenol lactone, and the PKA inhibitor, H-89. Additionally, the product of iPLA2 hydrolysis of plasmenylcholine and lysoplasmenylcholine elicited CREB phosphorylation in bovine pulmonary endothelial cells. Taken together, the present studies suggest that dsRNA as well as other agonists of endothelial cells elicit signaling mechanisms that include in part CREB phosphorylation mediated by iPLA2.

Reactive chlorinating species produced during neutrophil activation target tissue plasmalogens: production of the chemoattractant, 2-chlorohexadecanal.

Recently alpha-chloro fatty aldehydes have been shown to be products of reactive chlorinating species targeting the vinyl ether bond of plasmalogens utilizing a cell-free system. Accordingly, the present experiments were designed to show that alpha-chloro fatty aldehydes are produced by activated neutrophils and to determine their physiologic effects. A sensitive gas chromatography-mass spectrometry technique was developed to detect pentafluorobenzyl oximes of alpha-chloro fatty aldehydes utilizing negative ion chemical ionization. Phorbol 12-myristate 13-acetate activation of neutrophils resulted in the production of both 2-chlorohexadecanal and 2- chlorooctadecanal through a myeloperoxidase-dependent mechanism that likely involved the targeting of both 16 and 18 carbon vinyl ether-linked aliphatic groups present in the sn-1 position of neutrophil plasmalogens. 2-Chlorohexadecanal was also produced by fMLP-treated neutrophils. Additionally, reactive chlorinating species released from activated neutrophils targeted endothelial cell plasmalogens resulting in 2-chlorohexadecanal production. Physiologically relevant concentrations of 2-chlorohexadecanal induced neutrophil chemotaxis in vitro suggesting that alpha-chloro fatty aldehydes may have a role in neutrophil recruitment. Taken together, these studies demonstrate for the first time a novel biochemical mechanism that targets the vinyl ether bond of plasmalogens during neutrophil activation resulting in the production of alpha-chloro fatty aldehydes that may enhance the recruitment of neutrophils to areas of active inflammation.

220- and 130-kDa MLCKs have distinct tissue distributions and intracellular localization patterns.

To better understand the distinct functional roles of the 220- and 130-kDa forms of myosin light chain kinase (MLCK), expression and intracellular localization were determined during development and in adult mouse tissues. Northern blot, Western blot, and histochemical studies show that the 220-kDa MLCK is widely expressed during development as well as in several adult smooth muscle and nonmuscle tissues. The 130-kDa MLCK is highly expressed in all adult tissues examined and is also detectable during embryonic development. Colocalization studies examining the distribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDa MLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB or F-actin. In contrast, the 220-kDa MLCK did not colocalize with either nonmuscle myosin II isoform but instead colocalizes with thick interconnected bundles of F-actin. These results suggest that in vivo, the physiological functions of the 220- and 130-kDa MLCKs are likely to be regulated by their intracellular trafficking and distribution.

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts.

Thus far, determining the relative contribution of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and Ca2+-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca2+, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 +/- 0.02 mol PO4/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 +/- 0.05 and 0.82 +/- 0.05 mol PO4/mol RLC, respectively, within 2.5 min. Ca2+ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells.