Diphosphorylation Of Myosin Light Chain Research Articles

Critical role of Rho proteins in myosin light chain di-phosphorylation during early phase of endothelial barrier disruption

We previously reported the Rho-associated coiled-coil containing protein kinase (ROCK)-mediated di-phosphorylation of myosin light chain (MLC) and actin bundle formation at the cell periphery as early events of the endothelial barrier disruption. We herein examined the role of RhoA during early events of barrier disruption. Treatment of cultured porcine aortic endothelial cells with simvastatin prevented the decrease in trans-endothelial electrical resistance, MLC di-phosphorylation and peripheral actin bundle formation seen 3 min after thrombin stimulation. Co-treatment with geranylgeranyl pyrophosphate rescued the thrombin-induced events. Thrombin increased a GTP-bound form of RhoA and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at the ROCK site. The intracellular introduction of the inhibitory protein of RhoA inhibited the thrombin-induced di-phosphorylation of MLC. However, knockdown of either one of RhoA, RhoB or RhoC failed to inhibit thrombin-induced MLC di-phosphorylation. The findings suggest that Rho proteins play a critical role during early events of thrombin-induced barrier disruption.

Uniaxial stretching device for studying maturity-dependent morphological response of epithelial cell monolayers to tensile strain

Immature cell monolayers are vulnerable to tensile strain, which can cause severe diseases including fibrosis, cancer, and atherosclerosis. However, little is known about how immature cell monolayers respond to tensile strain. To study the effect of tensile strain on the morphology of cells in immature monolayers, we developed a miniaturized stretching device that allows stable real-time imaging of changes in cell shape induced by uniaxial static stretching of up to 20%. Upon stretching, the shape of cells in the immature (2 day-cultured) monolayer changed dynamically, unlike in the mature (7 day-cultured) monolayer. Dynamic changes in the immature cell monolayers induced by tensile strain can be explained by the increase in di-phosphorylated myosin light chain (pp-MLC), which initiates actomyosin contraction. We suggest that our stretching-observation system can be used to investigate the morphological response of epithelial cell monolayers to tensile strain.

A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction.

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di‐phosphorylated myosin light chain (ppMLC)‐driven contraction of actomyosin‐based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC‐triggered system at TJ‐associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule‐associated proteins in the AJC‐enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ‐, but not at AJ‐, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ‐associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule‐facilitated manner. Our results uncovered a hitherto unknown microtubule‐LUZP1 association at TJ‐associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cells.

In previous work, we reported that plasma membrane potential depolarization (PMPD) provokes cortical F-actin remodeling in bovine corneal endothelial (BCE) cells in culture, which eventually leads to the appearance of intercellular gaps. In kidney epithelial cells it has been shown that PMPD determines an extracellular-signal-regulated kinase (ERK)/Rho-dependent increase in diphosphorylated myosin light chain (ppMLC). The present study investigated the signaling pathways involved in the response of BCE cells to PMPD. Differently to renal epithelial cells, we observed that PMPD leads to a decrease in monophosphorylated MLC (pMLC) without affecting diphosphorylated MLC. Also, that the pMLC reduction is a consequence of cyclic adenosine 3',5'-monophosphate (cAMP)/protein kinase A (PKA) activation. In addition, we found evidence that the cAMP increase mostly depends on soluble adenylyl cyclase activity. Inhibition of this enzyme reduces the effect of PMPD on the cAMP rise, F-actin remodeling, and pMLC decrease. No changes in phosho-ERK were observed, although we could determine that RhoA undergoes activation. Our results suggested that active RhoA is not involved in the intercellular gap formation. Overall, the findings of this study support the view that, differently to renal epithelial cells, in BCE cells PMPD determines cytoskeletal reorganization via activation of the cAMP/PKA pathway.

ZIPK mediates endothelial cell contraction through myosin light chain phosphorylation and is required for ischemic-reperfusion injury.

The paracellular gap formed by endothelial cell (EC) contraction is fundamental for endothelium permeability, but the mechanism underlying EC contraction has yet to be determined. Here, we identified the zipper-interacting protein kinase (ZIPK) as the kinase for EC contraction and myosin light chain (MLC) phosphorylation. Inhibition of ZIPK activity by pharmacological inhibitors and small interfering RNAs led to a significant decrease in the mono- and diphosphorylation of MLCs along with a contractile response to thrombin, suggesting an essential role of ZIPK in EC paracellular permeability. To assess the role of ZIPK in vivo, we established mouse lines with conditional deletion of Zipk gene. The endothelium-specific deletion of Zipk led to embryonic lethality, whereas the UBC-CreERT2-mediated deletion of Zipk by tamoxifen induction at adulthood caused no apparent phenotype. The induced deletion of Zipk significantly inhibited ischemia-reperfusion-induced blood-brain barrier dysfunction and neuronal injuries from middle cerebral artery occlusion and reperfusion, as evidenced by reduced infarct and edema volume, attenuated Evans blue dye leakage, and improved neuronal behavior. We thus concluded that ZIPK and its phosphorylation of MLC were required for EC contraction and ischemic neuronal injuries. ZIPK may be a prospective therapeutic target for stroke.-Zhang, Y., Zhang, C., Zhang, H., Zeng, W., Li, S., Chen, C., Song, X., Sun, J., Sun, Z., Cui, C., Cao, X., Zheng, L., Wang, P., Zhao, W., Zhang, Z., Xu, Y., Zhu, M., Chen, H. ZIPK mediates endothelial cell contraction through myosin light chain phosphorylation and is required for ischemic-reperfusion injury.

Asiatic acid stabilizes cytoskeletal proteins and prevents TNF-α-induced disorganization of cell-cell junctions in human aortic endothelial cells

Endothelial hyperpermeability represents an initiating step in early atherosclerosis and it often occurs as a result of endothelial barrier dysfunction. Asiatic acid, a major triterpene isolated from Centella asiatica (L.) Urban, has previously been demonstrated to protect against tumor necrosis factor (TNF)-α-induced endothelial barrier dysfunction. The present study aimed to investigate the mechanisms underlying the barrier protective effect of asiatic acid in human aortic endothelial cells (HAECs). The localization of F-actin, diphosphorylated myosin light chain (diphospho-MLC), adherens junctions (AJs) and tight junctions (TJs) was studied using immunocytochemistry techniques and confocal microscopy. Their total protein expressions were examined using western blot analysis. The endothelial permeability was assessed using In Vitro Vascular Permeability Assay kits. In addition, intracellular redistribution of the junctional proteins was evaluated using subcellular fractionation kits. We show that asiatic acid stabilized F-actin and diphospho-MLC at the cell periphery and prevented their rearrangement stimulated by TNF-α. However, asiatic acid failed to attenuate cytochalasin D-induced increased permeability. Besides, asiatic acid abrogated TNF-α-induced structural reorganization of vascular endothelial (VE)-cadherin and β-catenin by preserving their reticulum structures at cell-cell contact areas. In addition, asiatic acid also inhibited TNF-α-induced redistribution of occludin and zona occludens (ZO)-1 in different subcellular fractions. In conclusion, the barrier-stabilizing effect of asiatic acid might be associated with preservation of AJs and prevention of TJ redistribution caused by TNF-α. This study provides evidence to support the potential use of asiatic acid in the prevention of early atherosclerosis, which is initiated by endothelial barrier dysfunction.

Neural Signaling Regulates Cancer Cell Physical Phenotypes

The physical phenotypes of tumor cells are critical in metastasis. To disseminate from the primary tumor, cancer cells generate protrusive and contractile forces to move through the surrounding extracellular matrix. Once in circulation, the physical properties of tumor cells allow them to survive fluid shear stresses. Identifying factors that regulate physical properties of tumor cells will identify important points of therapeutic leverage to stop metastasis. Our recent studies found that neural signaling regulates tumor cell physical phenotypes. We identified that activation of β-adrenergic (βAR) signaling regulates the physical properties of breast cancer (MDA-MB-231-HM) cells through increased myosin II activity; this makes cells stiffer and more invasive. When cells are treated with the βAR agonist, isoproterenol, we find increased levels of diphosphorylated myosin light chain (ppMLC) at serine 19 and threonine 18, indicating increased myosin II activity. We also determine the impact of βAR-induced myosin II activity on tumor cell contractility, invasion, as well as viability after exposure to physiologically-relevant fluid shear stresses. Importantly, activation of myosin II by βAR signaling is blocked by β-blocker treatment; these widely used drugs are showing exciting potential to be repurposed for cancer treatment. Our findings demonstrate the neural regulation of tumor cell physical phenotypes and provide new mechanistic insight into how modulating βAR signaling can be an effective anti-metastasis strategy.

Solo and Keratin Filaments Regulate Epithelial Tubule Morphology.

Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.Key words: epithelial tubulogenesis, Solo, keratin, Rho-GEF, myosin.

Oxidized Low-Density Lipoprotein-Induced Cyclophilin A Secretion Requires ROCK-Dependent Diphosphorylation of Myosin Light Chain

Objective: Accumulation of cyclophilin A (CyPA) within atherosclerotic lesions is thought to be implicated in the progression of atherosclerosis. However, the source of CyPA within atherosclerotic lesions is still unknown. The aim of this study is to determine the role of oxidized low-density lipoproteins (ox-LDL) in vascular smooth muscle cell (VSMC)-derived CyPA secretion and the underlying mechanism. Methods and Results: Abundant CyPA and α-smooth muscle actin (α-SMA) expressed in atherosclerotic lesions was observed in apolipoprotein E-deficient mice. ox-LDL induced CyPA secretion from a primary culture of rat aortic smooth muscle cells in a dose- and time-dependent manner. Sulfosuccinimidyloleate, a CD36 inhibitor, prevented the ox-LDL-induced CyPA secretion. Pre-exposure to either the actin-depolymerizing agent cytochalasin D or the actin-polymerizing agent jasplakinolide inhibited CyPA secretion induced by ox-LDL. Gene silencing of vesicle-associated membrane protein 2 suppressed ox-LDL-induced CyPA secretion. ox-LDL caused the phosphorylation of myosin light chain (MLC). Inhibition of MLC by blebbistatin reversed the secretion of CyPA and the phosphorylation of MLC induced by ox-LDL. MLC kinase inhibitor ML-7 reduced the monophosphorylation of MLC but did not reduce CyPA secretion. Pretreatment with the rho-associated coiled-coil kinase (ROCK) inhibitor Y27632 blocked diphosphorylation of MLC and secretion of CyPA induced by ox-LDL. Conclusions: ox-LDL-induced CyPA secretion requires vesicle transportation, actin remodeling and ROCK-dependent diphosphorylation of MLC. VSMC-derived CyPA induced by ox-LDL may be associated with increased CyPA expression in atherosclerotic lesions.

Abstract WP438: Rho Kinase-mediated Di-phosphorylation of Myosin Light Chain in the Sub-membranous Regions and Circumferential Actomyosin Contraction Mediate Thrombin-induced Barrier Disruption in Vascular Endothelial Cells

The disruption of blood-brain barrier plays a critical role in the pathophysiology of cerebrovascular diseases. Thrombin is one of the major factors which cause barrier disruption. The phosphorylation of myosin light chain (MLC) is a key signal of barrier disruption. MLC is thought to be di-phosphorylated sequentially at Ser19 and then Thr18, thereby inducing stress fiber formation to generate traction force to disrupt cell-cell contact. However, it is unclear how the phosphorylation at two sites contributes to barrier disruption. The present study investigated the role of mono- and di-phosphorylation of MLC (MLC-P and MLC-PP) in thrombin-induced barrier disruption. Thrombin (1 u/mL) decreased the transendothelial electrical resistance (TEER) with a peak at 3-5 min in porcine aortic endothelial cells (PAEC). A Phos-tag SDS-PAGE method was used to quantify the amount of MLC-P and MLC-PP. PAEC at confluence contained 25% MLC-P and 2% MLC-PP before stimulation. Upon thrombin stimulation, MLC-P marginally increased, while MLC-PP transiently increased to a peak of 35% at 3-5 min. MLC-P was localized mainly in the peri-nuclear region, while MLC-PP was localized mainly in the sub-membranous region of cell-cell contact. MLC-PP was also co-localized with the peripheral actin bundles. In contrast, thrombin induced stress fiber formation and localization of MLC-P and MLC-PP on the stress fibers when the cell-cell contact was loosed by removing extracellular Ca 2+ or in the cells at the growing phase with sparse cell-cell contact. Two different Rho kinase inhibitors, Y27632 and H1152, inhibited the thrombin-induced increase in MLC-PP, sub-membranous localization of MLC-PP and decrease in TEER, while having no effect on the level of MLC-P. Inhibition of myosin ATPase activity by 100 μmol/L blebbistatin inhibited the thrombin-induced decrease in TEER. The present study suggests that MLC-P and MLC-PP are independently regulated in endothelial cells, and that Rho kinase-mediated MLC-PP in the sub-membranous regions and the circumferential, but not radial, contraction plays a critical role in the thrombin-induced barrier disruption. Inhibition of MLC-PP thus provides a crucial strategy for restoring normal function of the blood-brain barrier.

Abstract 9614: Ca 2+ -Independent Di-phosphorylation of Myosin Light Chain by Rho-associated Coiled-Coil Protein Kinase (ROCK) in the Subjunctional Regions of Cell-Cell Contact Mediates Thrombin-Induced Barrier Dysfunction in Vascular Endothelial Cells

A disruption of endothelial barrier function is a predisposing factor for the development of atherosclerosis. The phosphorylation of myosin light chain (MLC) is a key signal of barrier dysfunction. MLC phosphorylation is thought to take place sequentially at S19 and then T18, induces actin stress fiber formation and generates traction force to disrupt cell-cell contact. However, it is unclear how the phosphorylation at two sites contributes to barrier dysfunction. The present study investigated the role of mono- and di-phosphorylation of MLC (MLC-P and MLC-PP) in thrombin-induced barrier dysfunction. Thrombin (1 u/mL) increased the cytosolic Ca 2+ concentration and decreased the transendothelial electrical resistance (TEER) with a peak at 1 and 3-5 min, respectively, in porcine aortic endothelial cells (PAEC). A new SDS-PAGE method using a Phos-tag TM compound, which specifically binds to a phosphate group, allowed separate quantification of MLC-P and MLC-PP. PAEC at confluence contained 25% MLC-P and 2% MLC-PP under resting conditions. Thrombin stimulation only slightly increased MLC-P at 3 min, while it showed up on stress fibers. MLC-PP increased to a peak of 35% at 3-5 min, and thereafter declined to the resting level within 15 min. MLC-PP was localized in the subjunctional region of cell-cell contact in addition to the stress fibers at 3 min. ROCK inhibitors, Y27632 and H1152, inhibited the thrombin-induced decrease in TEER, the increase in MLC-PP and the subjunctional localization of MLC-PP, while having no effect on the Ca 2+ response. The MLC-P level remained unaffected by Y27632, while the stress fiber localization was inhibited. Gαq inhibitor, YM254890, inhibited the Ca 2+ response and the decrease in TEER, but it had no effect on the increase in MLC-PP. The introduction of dominant negative mutants of Gα12, Gα13 and Gαq using a cell-penetrating peptide inhibited the thrombin-induced decrease in TEER. The present study provides the first evidence that MLC-P and MLC-PP are differentially regulated in endothelial cells, and that Ca 2+ -independent, ROCK-mediated MLC-PP in the subjunctional regions plays a critical role in the thrombin-induced barrier dysfunction. Inhibition of MLC-PP is thus necessary to restore endothelial barrier dysfunction.

Vascular smooth muscle myosin light chain diphosphorylation: Mechanism, function, and pathological implications

Smooth muscle contraction is activated primarily by phosphorylation at S19 of the 20-kDa regulatory light chain subunits of myosin II (LC(20) ) catalyzed by Ca(2+) /calmodulin-dependent myosin light chain kinase. Other kinases, for example, integrin-linked kinase (ILK), Rho-associated kinase (ROCK), and zipper-interacting protein kinase (ZIPK), can phosphorylate T18 in addition to S19, which increases the actin-activated myosin MgATPase activity at subsaturating actin concentrations ∼3-fold. These phosphorylatable residues and the amino acid sequence surrounding them are highly conserved throughout the animal kingdom; they are also found in an LC(20) homolog within the genome of Monosiga brevicollis, the closest living relative of metazoans. LC(20) diphosphorylation has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli and in pathophysiological situations associated with hypercontractility. LC(20) diphosphorylation has also been observed frequently in cultured cells where it activates force generation. Kinases such as ILK, ROCK, and ZIPK, therefore, are potential therapeutic targets in the treatment of, for example, cerebral vasospasm following subarachnoid hemorrhage and atherosclerosis.

Enhanced contractility and myosin phosphorylation induced by Ca2+-independent MLCK activity in hypertensive rats

The role of Ca(2+) sensitization induced by a Ca(2+)-independent myosin light chain kinase (MLCK) in hypertension has not been determined. The aim of this study was to clarify the role of possible Ca(2+)-independent MLCK activity in hypertension. We compared increases in contractile force and phosphorylation of myosin light chain (MLC) evoked by calyculin A, a phosphatase inhibitor, in β-escin-permeabilized mesenteric arteries at pCa 9.0 between spontaneously hypertensive rat (SHR) and Wistar Kyoto rat (WKY). We found that there was no detectable phosphorylation of MLC at pCa 9.0, but that the administration of 1 μM calyculin A gradually increased force and mono- and di-phosphorylation of MLC. This contraction was inhibited by staurosporine but not by wortmannin, Y-27632, or calphostin-C. The calyculin A-induced contraction was significantly greater in the SHR than in the WKY and was associated with an increase in mono- and di-phosphorylation of MLC. SM-1, a zipper-interacting protein kinase (ZIPK)-inhibiting peptide, significantly inhibited the amplitude of the calyculin A-induced contraction and di-phosphorylation. Total ZIPK expression (54 + 32 kDa) was greater in the SHR than in the WKY. Phosphorylation of myosin phosphatase target subunit at Thr(697), but not at Thr(855), was consistently stronger in the SHR than in the WKY in calyculin A-treated tissues at pCa 9.0. Our results suggest that Ca(2+)-independent MLCK activity is enhanced in the SHR, and that ZIPK plays, at least in part, an important role as a candidate for this kinase in rat mesenteric arteries.

Role of Caldesmon in hypoxia‐mediated endothelial cell response

Pulmonary hypertension (PH) involves stress‐mediated pathways producing endothelial and smooth muscle cell dysfunction. The actin cytoskeleton is important in vascular remodeling and our earlier studies strongly suggested the role of caldesmon (Cald1) in endothelial cytoskeleton remodeling. Cald1 exhibits 2 isoforms: h‐Cald1 found in smooth muscle and l‐Cald1, expressed in non‐muscle cells and involved in cell proliferation and contractility. Both Cald1 isoforms are important in hypoxia‐induced PH and Cald1 surfaced as a candidate gene in 2 rodent PH therapy trials (simvastatin and sorafenib, Sor). We hypothesized that l‐Cald1 is an important regulator of vascular remodeling in PH. We validated Sor array data with increased l‐ and h‐ Cald1 protein levels in lung homgenates from hypoxia‐(1.5‐ & 2.5‐fold) and in hypoxia/SU5416 (H‐SU)‐ challenged rats (2‐ & 4‐fold compared to normoxia at 3.5 wks). Sor treatment reduced l‐ Cald1 levels in H‐SU while h‐ Cald1 remained elevated. Given isoform‐specific changes, we examined l‐Cald1 in vitro in HLMECs exposed to hypoxia (30 min‐4h). Gene and protein l‐Cald1 levels were increased at 1h (2.5‐ & 1.8‐fold) and 4h (1.6‐fold). Diphosphorylated myosin light chain (dpMLC) levels increased (2‐fold) by 30 min and decreased (1.5‐fold) by 2h with no change in total MLC. Thus, l‐Cald is potentially a key regulator in vascular remodeling in PH. NIH support: F32 HL090359‐01

Calyculin A retraction of mature megakaryocytes proplatelets from embryonic stem cells

Platelets are produced by megakaryocytes (MKs) through proplatelet formation (PPF), or cytoplasmic extensions, in vitro. Through the use of video-enhanced light microscopy, as well as localization of cytoskeletal proteins by confocal microscopy, the reaction of fully mature MK proplatelets, derived from murine embryonic stem cells, to various agents was studied. Calyculin A (protein phosphatase 1/2A inhibitor) treatment induced proplatelet retraction. In MKs with PPF, the expression of actin, myosin IIA, monophosphorylated myosin light chain (MLC-P1), and diphosphorylated myosin light chain (MLC-P2) was diffusely located. Following calyculin A treatment, actin was diffusely localized in retracted MKs and was expressed particularly in the periphery. MLC-P1 was also localized primarily in the periphery; however, MLC-P2 was expressed mostly in the inner area of proplatelets. Protein phosphatase inhibitors may result in increased hyperphosphorylation of localized MLC, which could alter the balance of actomyosin force in a cell, and therefore induce proplatelets retraction.

Mammalian SEPT2 Is Required for Scaffolding Nonmuscle Myosin II and Its Kinases

Mammalian septin SEPT2 belongs to a conserved family of filamentous GTPases that are associated with actin stress fibers in interphase cells and the contractile ring in dividing cells. Although SEPT2 is essential for cytokinesis, its role in this process remains undefined. Here, we report that SEPT2 directly binds nonmuscle myosin II (myosin II), and this association is important for fully activating myosin II in interphase and dividing cells. Inhibition of the SEPT2-myosin II interaction in interphase cells results in loss of stress fibers, while in dividing cells this causes instability of the ingressed cleavage furrow and dissociation of the myosin II from the Rho-activated myosin kinases ROCK and citron kinase. We propose that SEPT2-containing filaments provide a molecular platform for myosin II and its kinases to ensure the full activation of myosin II that is necessary for the final stages of cytokinesis.

Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy

BackgroundIn anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells.ResultsParaformaldehyde fixation and Triton X-100 permeabilization preserved cell morphology and myosin light chain phosphorylation better than the alternative fixation/permeabilization methods tested. We utilized automated microscopy methods to acquire three color images, determine cell spread area, and quantify the intensity of staining within each cell with anti-phospho-MLC antibody. Our results indicate that A10 rat aortic smooth muscle cells exhibit a re producible non-Gaussian distribution of MLC phosphorylation across a population of unsynchronized genetically identical cells. Adding an inhibitor of Rho kinase, Y27632, or plating cells on a low density of fibronectin, reduced phospho-myosin light chain signal as expected. On the other hand, adding calyculin A, an activator of contractility, increased myosin light chain phosphorylation. The IC50 for myosin light chain phosphorylation using Y27632 was determined to be 2.1 ± 0.6 micrometers. We observed a positive linear relationship between cell area and myosin light chain diphosphorylation, which is consistent with what has been reported in the literature using other methods.ConclusionOur results show that using proper specimen fixation techniques and background subtraction methods, imaging cytometry can be used to reliably measure relative myosin light chain phosphorylation in individual adherent cells. Importantly, the ability to make this measurement in adherent cells allows for simultaneous measurement of and correlation with other parameters of cellular topography such as morphology and cell-cell proximity. This assay has potential application in screening for drug development.

Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery

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-MLCK- and myosin-binding site in SV, SV1-171, rearranges and co-localizes with mono- and di-phosphorylated myosin light chain and with L-MLCK, but not with the short form of MLCK (S-MLCK) or with myosin phosphatase. Thus, the membrane protein SV apparently contributes to myosin II assembly during cell spreading by modulating myosin II regulation by L-MLCK.

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH(2) terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 mug/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.

Ca2+-Independent, Inhibitory Effects of Cyclic Adenosine 5′-Monophosphate on Ca2+ Regulation of Phosphoinositide 3-Kinase C2α, Rho, and Myosin Phosphatase in Vascular Smooth Muscle

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca(2+)-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca(2+)-induced Ca(2+)-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca(2+)-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca(2+) mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca(2+)-mediated Rho activation is dependent on class II alpha-isoform of phosphoinositide 3-kinase (PI3K-C2alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) in manners dependent on both PI3K and Rho kinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca(2+)-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2alpha, Rho, and Rho kinase in a manner independent of Ca(2+) and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.