Abstract. The phosphorylation of regulatory myosin light chains by the Ca2÷/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B., and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. . We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2-to 2.5-fold within 5 min and remains elevated for at least 60 min.The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation.Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F-actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.NDOTHELIAL cells lining most vessels form a continuous layer that normally constrains proteins as well as formed blood elements to the vascular lumen. The loss of continuity of the endothelial sheet leads to increased permeability and the development of edema (30,47,51,58,71,75). The increase in microvascular permeability by inflammatory and chemical mediators has been correlated with the presence of small gaps between adjacent endothelial cells. Furthermore, mediators that disrupt actin filaments and chelate extracellular calcium have been implicated in changes in macromolecular flux. The factors that may play a role in altering the barrier function of the endothelium leading to edema include: (a) loss of Address all correspondence t...

Abstract. We have used an isometric force transducer to study contraction of two types of nonmuscle cells in tissue culture . This method permits the quantitative measurement of contractile force generated by cells of defined type under the influence of external agents while allowing detailed morphological observation . Chick embryo fibroblasts (CEF), which form a contractile network inside a collagen matrix, and human umbilical vein endothelial cells (HUVE), which are located in a monolayer on the surface of the collagen matrix, were studied . CEF and HUVE in 10% FCS produce a substantial tension of 4.5 ± 0.2 x 104 dynes/cm2 and 6.1 x 104 dynes/cm2, respectively. Both M MST of what is known about force generation in animal cells has come from studies of muscle. However, the generation of force is essential to such nonmuscle cell functions as wound contraction, gastrulation, and connective tissue morphogenesis . We have developed a simple quantitative technique to study isometric contraction of cells in tissue culture . This technique allows mechanical study of a variety ofnonmuscle and smooth muscle cells undergoing biochemical and genetic manipulations .Nonmuscle cells are believed to generate force through interaction between actin and myosin as in smooth and skeletal muscle (1,20,25,26) . Recently it has been demonstrated that nonmuscle cells express isoforms ofnonsarcomeric myosin distinct from those expressed in smooth muscle (18,19) . The nature of these isoforms has been characterized ; however, their function remains unclear. Thus, a quantitative study of isometric contraction in a tissue culture system may provide insight into the mechanical specialization of nonmuscle myosin isoforms.Unlike skeletal muscle, in which the cytoskeleton is arranged in a well-defined, stable organization, nonmuscle cells rearrange their cytoskeletons in response to physiological stimuli such as growth factors and secretagogues. It is clear that studies correlating cell mechanics with cytoskeletal morphology and biochemistry are needed to understand mechanisms through which nonmuscle cells control their mechanical machinery. In this study, we present the results of initial efforts to assay quantitatively the force produced in cell types contract when stimulated with thrombin, generating a force per cell cross-sectional area of -105 dynes/cm2, a value approximately an order of magnitude less than smooth muscle . The integrity of the actin cytoskeleton is essential for force generation, as disruption of actin microfilaments with cytochalasin D results in a rapid disappearance of force . Intact microtubules appear to reduce isometric force exerted by CEF, as microtubule-disrupting drugs result in increased tension . Contraction by HUVE precedes a dramatic rearrangement of actin microfilaments from a circumferential ring to stress fibers .vitro by nonmuscle cells as they undergo stimulation and morphological change. We present measurements of the isometric force developed by a contractile network ofchick embryo fibroblasts (CEF...

Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca2+. Retraction Skinned preparations of smooth muscle in which the membrane barrier to influx of large molecules and charged small molecules has been destroyed have been important in analyzing the biochemical characteristics of the contractile process in these cells (6,7,9). Similar preparations offibroblasts (10,11) and epithelial cells (12, 13) have also been studied. We have developed a preparation of permeabilized ECs that has provided us with the opportunity to investigate the role of ATP, Ca2+, calmodulin, and MLCK in the initiation of EC retraction.EXPERIMENTAL PROCEDURES Cell Culture. The bovine pulmonary artery cell line developed by Del Vecchio and Smith (14) was obtained from American Type Culture Collection (CLL-209). Cells were grown in Eagle's minimal essential medium supplemented with 2 mM glutamine, 10o or 20%o fetal calf serum, penicillin at 50 units/ml, and streptomycin at 50 gg/ml. Cells were maintained at 370C in a humidified 5% C02/95% air atmosphere. Cells used in these studies were 7 days postconfluent (2).Cell Permeabilization. EC cultures were washed with Dulbecco's phosphate-buffered saline (DPBS) (pH 7.2) and permeabilized with 2 ml of buffer A (20 mM Pipes/10 mM imidazole/50 mM KCI/1 mM EGTA/1 mM MgSO4/0.2 mM dithiothreitol/5 ,g of aprotonin per ml/5 ug of leupeptin per ml/10 ,ug of soybean trypsin inhibitor per ml/0.1 mM phenylmethylsulfonyl fluoride/0.5 mM benzamidine, pH 6.5) containing 25 ,ug of saponin per ml, incubated at 37°C for 10 min, and washed with 2 ml of buffer A without saponin. Care was taken not to allow the permeabilized monolayers to dry.Permeabilized

Recently ␣-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 ␣-chloro fatty aldehydes are produced by activated neutrophils and to determine their physiologic effects. A sensitive gas chromatographymass spectrometry technique was developed to detect pentafluorobenzyl oximes of ␣-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 fMLPtreated 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 ␣-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 ␣-chloro fatty aldehydes that may enhance the recruitment of neutrophils to areas of active inflammation.

Phosphorylation of myosin II regulatory light chains (RLC) by Ca2؉ /calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Posttranslational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPasedependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 ؎ 0.21 mol of PO 4 /mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.The PAK 1 family of serine/threonine protein kinases have been implicated in a broad spectrum of signal transduction pathways leading to diverse physiological end points, including cytoskeletal reorganization, apoptosis, and Ras-mediated cell transformation (1, 2). All PAK isoforms are direct effectors of the Rho family GTP-binding proteins Rac and Cdc42, suggesting that cell-specific responses arise from either selective regulation of G protein activation in response to unique agonists or selective phosphorylation of tissue-specific protein kinase substrates.In the initial studies of the relative roles of G protein activation and protein kinase activity in actin reorganization, Sells et al. (3) demonstrated that microinjection of activated PAK1 induced rapid formation of polarized filopodia in Swiss 3T3 cells. However, the relative importance of PAK-dependent phosphorylation of unique substrates during Cdc42/Racdependent cytoskeletal reorganization is incompletely understood. Transient transfection of HeLa cells with constitutively active PAK1 promoted cytoskeletal rearrangement, which was entirely analogous to that observed in Cdc42 and Rac transfected cells (4). In subsequent studies, a peptide that inhibited kinase activity in PAK blocked Cdc42, Rac, and constitutively active PAK-induced morphological changes in transfected HeLa cells (5). An important role for PAK-mediated phosphorylation in actin reorganization has also been supported by studies in permeabilized (6) and intact (7) endothelial cells as well as skinned smooth muscle cells (8). Permeabilized endothelial cells incubated with Cdc42-activated PAK2 or the catalytic domain of PAK2 underwent ATP-dependent retraction and actin reorganization (6). In addition, these studies using purified enzymes established that the regulatory nonmuscle and smooth muscle myosin II light chain is a substrate for PAK2 (9, 1...

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.

SummaryThe 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.

The mechanism or mechanisms by which polymorphonuclear leukocytes (PMN) penetrate junctions between neighboring endothelial cells (EC) to traverse endothelial barriers remain unresolved. We report that chemoattractant-stimulated PMN induce a coordinate increase in both phosphorylation of serine 19 and threonine 18 of EC myosin regulatory light chains and isometric tension generation by EC monolayers. Unstimulated PMN had no effect on either parameter. These findings, coupled with our previous report (Huang et al., J. Cell Biol. 120: 1371-1380, 1993) that chemoattractant-stimulated PMN cause a rise in EC cytosolic free Ca2+, provide strong presumptive evidence that myosin light chain kinase is the EC enzyme responsible for initiating myosin light chain phosphorylation, EC contraction, and isometric tension generation in response to chemoattractant-stimulated PMN. We suggest that, by inducing phosphorylation of EC cytoskeletal proteins, chemoattractant-stimulated PMN induce EC to open their intercellular junctions, thereby facilitating transendothelial movement of these leukocytes.