Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization

Endothelial repair to reestablish structural integrity following wounding is a complex process. Since the actin cytoskeleton undergoes specific changes in distribution as quiescent endothelial cells switch to activated migrating cells over a 6-h period following wounding (Lee et al. 1996), we studied tyrosine phosphorylation in association with actin microfilaments and adhesion proteins using double immunofluorescent confocal microscopy. We showed that in a confluent monolayer phosphotyrosine localized at the periphery of the cell at vinculin cell-cell adhesion sites within the actin-dense peripheral band (DPB) and centrally at talin/vinculin cell-substratum adhesion sites at the ends of central microfilaments. Over a period of 6 h following in vitro wounding there was a reduction of peripheral phosphotyrosine associated with the loss of both cell-cell adhesion sites and the DPB (stage I). Concomitantly, an increase in central phosphotyrosine was associated with an increase in cell-substratum adhesion sites and central microfilaments parallel to the wound edge (stage II), which subsequently redistributed perpendicular to the wound edge (stage III). We also localized FAK and paxillin at the ends of parallel and perpendicular central microfilaments. Immunoprecipitation of paxillin showed increased phosphotyrosine and protein levels when prominent central microfilaments were present and underwent remodeling. Inhibition of tyrosine kinases by genistein and tyrosine phosphatases by sodium orthovanadate resulted in reduced endothelial repair associated with disruption of adhesion site formation and central microfilament formation/redistribution in each stage of repair. We suggest that tyrosine phosphorylation of adhesion proteins, such as paxillin, may be important in regulating the early stages of endothelial wound repair. Received: 22 March 1999 / Accepted: 24 March 1999     

Postnatal reorganization of actin filaments and differentiation of intercellular boundaries in the rat aortic endothelial cells

Postnatal change in the distribution of actin filaments in endothelial cells was studied in the rat aorta by use of rhodamine-phalloidin staining and confocal laser scanning microscopy. Endothelial cells of the rat aorta possessed two populations of actin filament bundles, namely, peripheral bands at the cell border and stress fibers running longitudinally in the cytoplasm. Aortic endothelial cells of the neonatal rat contained only stress fibers, whereas those of the 10-day-old rat developed both peripheral bands and stress fibers. After 20 days of age, aortic endothelial cells had predominantly peripheral bands with occasional stress fibers around the branch orifices. During postnatal development the length density of stress fibers in aortic endothelial cells decreased, whereas individual stress fibers in endothelial cells were shortened. Electron-microscopic observation revealed that the high intercellular boundaries of aortic endothelial cells at birth decreased in height and developed cytoplasmic interdigitations after 20 days of age. The occurrence of peripheral bands at the cell border is thought to be closely related to formation of cytoplasmic interdigitation which strengthens the mechanical connection between endothelial cells against increasing transmural pressure. Expression of stress fibers in aortic endothelial cells of the neonatal rat is supposed to be affected by longitudinal elongation of the developing aorta, whereas their postnatal decrease is though to be correlated with the change of fluid shear stress loaded in the aortic endothelium.     

Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells

Botulinum C2 toxin ADP-ribosylates actin in [32P]orthophosphate-labelled intact chick embryo cells (CEC). The toxin-induced rounding up of CEC is correlated with ADP-ribosylation of actin in intact cells in a time and concentration-dependent manner. Both, rounding up of cells and actin ADP-ribosylation, depend on the presence of both components of botulinum C2 toxin (components I and II) and are independent of the ability of CEC to divide. Treatment of CEC with botulinum C2 toxin induced a time-dependent disorganization of the typical architecture of the microfilament network as shown by fluorescein-phalloidin staining. Botulinum C2 toxin decreased the amount of Triton X-100 insoluble actin, while the fraction of Triton soluble actin was increased. Actin, which was 32P-labelled by botulinum C2 toxin in intact CEC, was recovered in the Triton soluble but not in the Triton insoluble actin fraction. It is suggested that in intact CEC botulinum C2 toxin causes ADP-ribosylation of G-actin but not of F-actin thereby leading to an accumulation in the pool of monomeric actin.     

Cryoinjury in endothelial cell monolayers

Developing successful cryopreservation strategies for corneas have proven to be more difficult than anticipated, because of the resulting loss of viability and detachment of endothelial cells from Descemet's membrane following cryopreservation of corneas. The objectives of this study are to develop a more detailed understanding of cryoinjury in human corneal endothelial cell (HCEC) monolayers and to examine the effects of storage temperature, cryoprotectant type and concentration, and cooling/warming rates on HCEC monolayers. Monolayers of endothelial cells attached to collagen-coated glass, immersed in an experimental solution (with and without cryoprotectant) were cooled at 1 degrees C/min to various temperatures (-5 to -40 degrees C), then thawed directly or cooled rapidly to -196 or to -80 degrees C before thawing. Cryoprotectants used were dimethyl sulfoxide and propylene glycol in concentrations of 1 and 2M. Monolayers were assessed for membrane integrity and detachment using SYTO/ethidium bromide fluorescent stain. The presence of cryoprotectants resulted in high recovery of membrane integrity and low monolayer detachment in monolayers thawed directly from temperatures down to -40 degrees C. In contrast, there was excessive detachment and loss of membrane integrity in monolayers cooled to -196 degrees C compared to monolayers cooled to -80 degrees C. Also, increasing cryoprotectant concentrations did not improve recovery of the monolayers. The higher recovery and lower detachment after storage at -80 degrees C compared to storage at -196 degrees C suggest that storage temperatures for corneas should be re-evaluated.     

Modeling actin filament reorganization in endothelial cells subjected to cyclic stretch

Hemodynamic forces affect endothelial cell morphology and function. In particular, circumferential cyclic stretch of blood vessels, due to pressure changes during the cardiac cycle, is known to affect the endothelial cell shape, mediating the alignment of the cells in the direction perpendicular to stretch. This change in cell shape proceeds a drastic reorganization at the internal level. The cellular scaffolding, mainly composed of actin filaments, reorganize in the direction which later becomes the cell’s long axis. How this external mechanical stimulus is ’sensed’ and transduced into the cell is still unknown. Here, we develop a mathematical model depicting the dynamics of actin filaments, and the influence of the cyclic stretch of the substratum based on the experimental evidence that external stimuli may be transduced inside the cell via transmembrane proteins which are coupled with actin filaments on the cytoplasmic side. Based on this view, we investigate two approaches describing the formulation of the transduction mechanisms involving the coupling between filaments and the membrane proteins. As a result, we find that the mechanical stimulus could cause the experimentally observed reorganization of the entire cytoskeleton simply by altering the dynamics of the filaments connected with the integral membrane proteins, as described in our model. Comparison of our results with previous studies of cytoskeletal dynamics reveals that the cytoskeleton, which, in the absence of the effect of stretch would maintain its isotropic distribution, slowly aligns with the precise direction set by the external stimulus. It is found that even a feeble stimulus, coupled with a strong internal dynamics, is sufficient to align actin filaments perpendicular to the direction of stretch.     

Cytoarchitecture of Kirsten sarcoma virus-transformed rat kidney fibroblasts: butyrate-induced reorganization within the actin microfilament network

Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading.     

Collective cell motion in endothelial monolayers

Collective cell motility is an important aspect of several developmental and pathophysiological processes. Despite its importance, the mechanisms that allow cells to be both motile and adhere to one another are poorly understood. In this study we establish statistical properties of the random streaming behavior of endothelial monolayer cultures. To understand the reported empirical findings, we expand the widely used cellular Potts model to include active cell motility. For spontaneous directed motility we assume a positive feedback between cell displacements and cell polarity. The resulting model is studied with computer simulations and is shown to exhibit behavior compatible with experimental findings. In particular, in monolayer cultures both the speed and persistence of cell motion decreases, transient cell chains move together as groups and velocity correlations extend over several cell diameters. As active cell motility is ubiquitous both in vitro and in vivo, our model is expected to be a generally applicable representation of cellular behavior.     

Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components

Microtubules and actin filaments regulate plasma membrane topography, but their role in compartmentation of caveolae-resident signaling components, in particular G protein-coupled receptors (GPCR) and their stimulation of cAMP production, has not been defined. We hypothesized that the microtubular and actin cytoskeletons influence the expression and function of lipid rafts/caveolae, thereby regulating the distribution of GPCR signaling components that promote cAMP formation. Depolymerization of microtubules with colchicine (Colch) or actin microfilaments with cytochalasin D (CD) dramatically reduced the amount of caveolin-3 in buoyant (sucrose density) fractions of adult rat cardiac myocytes. Colch or CD treatment led to the exclusion of caveolin-1, caveolin-2, beta1-adrenergic receptors (beta1-AR), beta2-AR, Galpha(s), and adenylyl cyclase (AC)5/6 from buoyant fractions, decreasing AC5/6 and tyrosine-phosphorylated caveolin-1 in caveolin-1 immunoprecipitates but in parallel increased isoproterenol (beta-AR agonist)-stimulated cAMP production. Incubation with Colch decreased co-localization (by immunofluorescence microscopy) of caveolin-3 and alpha-tubulin; both Colch and CD decreased co-localization of caveolin-3 and filamin (an F-actin cross-linking protein), decreased phosphorylation of caveolin-1, Src, and p38 MAPK, and reduced the number of caveolae/mum of sarcolemma (determined by electron microscopy). Treatment of S49 T-lymphoma cells (which possess lipid rafts but lack caveolae) with CD or Colch redistributed a lipid raft marker (linker for activation of T cells (LAT)) and Galpha(s) from lipid raft domains. We conclude that microtubules and actin filaments restrict cAMP formation by regulating the localization and interaction of GPCR-G(s)-AC in lipid rafts/caveolae.     

JNK and PI3K differentially regulate MMP-2 and MT1-MMP mRNA and protein in response to actin cytoskeleton reorganization in endothelial cells

Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK-dependent signaling pathway in endothelial cells. The pharmacological actin depolymerization agent cytochalasin D increased expression of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c-Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of phosphoinositide 3-kinase (PI3K), but not JNK, significantly decreased the amount of active MMP-2 following cytochalasin D stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF-induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF-induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signaling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression, whereas PI3K regulates protein levels of both MMP-2 and MT1-MMP. angiogenesis; mechanotransduction; vascular endothelial growth factor; c-Jun; phosphoinositide 3-kinase; membrane type 1-matrix metalloproteinase     

Microtubules, but not actin microfilaments, regulate vacuole motility and morphology in hyphae of Pisolithus tinctorius

While it is now recognised that transport within the endomembrane system may occur via membranous tubules, spatial regulation of this process is poorly understood. We have investigated the role of the cytoskeleton in regulating the motility and morphology of the motile vacuole system in hyphae of the fungus Pisolithus tinctorius by studying (1) the effects of anti-microtubule (oryzalin, nocodazole) and anti-actin drugs (cytochalasins, latrunculin) on vacuolar activity, monitored by fluorescence microscopy of living cells; and (2) the ultrastructural relationship of microtubules, actin microfilaments, and vacuoles in hyphae prepared by rapid-freezing and freeze-substitution. Anti-microtubule drugs reduced the tubular component of the vacuole system in a dose-dependent and reversible manner, the extent of which correlated strongly with the degree of disruption of the microtubule network (monitored by immunofluorescence microscopy). The highest doses of anti-microtubule drugs completely eliminated tubular vacuoles, and only spherical vacuoles were observed. In contrast, anti-actin drugs did not reduce the frequency of tubular vacuoles or the motility of these vacuoles, even though immunofluorescence microscopy confirmed perturbation of microfilament organisation. Electron microscopy showed that vacuoles were always accompanied by microtubules. Bundles of microtubules were found running in parallel along the length of tubular vacuoles and individual microtubules were often within one microtubule diameter of a vacuole membrane. Our results strongly support a role for microtubules, but not actin microfilaments, in the spatial regulation of vacuole motility and morphology in fungal hyphae.     

Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair cells.     

Actin microfilament dynamics and actin side-binding proteins in plants

Actin microfilaments are highly organized and essential intracellular components of organelle movement and cell morphogenesis in plants. The organization of these microfilaments undergoes dynamic changes during cell division, elongation, and differentiation. Recent live-cell imaging of plant actin microfilaments has revealed their native organization and remarkable dynamics. In addition, characterization of plant actin side-binding proteins has progressed rapidly by genetic, biochemical, and bioinformatic approaches. The gathering and integration of microscopy-based information from actin microfilament dynamics and the molecular identification of actin side-binding proteins have provided considerable insights into actin microfilament-dependent events and actin microfilament organization in plants.     

Zeta-potentials of intact cell monolayers determined by electro-osmosis

Summary By measuring the electro-osmotic flow velocity of buffer through a glass capillary, on the inner surface of which cells have been grown, the ζ-potential of these cells can be determined without the changes in ζ-potential, viability and morphology caused by the chemical, enzymatic or mechanical pretreatment hitherto necessary for cell dispersion in the microelectrophoretic method. Cells grown in a monolayer inside the capillaries were: BGM, HEP-2, and RPMI 1846 (the last one also could be grown in suspension). R. M. F. was supported by NIH training grant AI00442.     

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.     

Permeability characteristics of cultured endothelial cell monolayers

The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.     

Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis

Background  

Plant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs) and actin microfilaments (MFs). Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown.     

The distribution of microfilament bundles in rabbit endothelial cells in the intact aorta and during wound healing in situ

The distribution of microfilament (MF) bundles in rabbit thoracic aortic endothelial cells (EC) fixed in situ was examined using en face preparations and the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In the normal aorta, prominent peripheral MF bundles are seen near the cell borders running the full length of each cell, parallel to the direction of blood flow, while shorter less prominent bundles are seen in the more central regions. In EC covering the flow dividers at intercostal ostia, the central MF bundles are more prominent, longer, and more numerous than in the other regions of the aorta examined. This increase in the number, size, and length of central MF bundles may result from the response of the cells to the higher shear forces present in this region of the vessel wall. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, there is an initial reduction in the size of all of the MF bundles in cells near the wound edge. This is followed by an increase in the number and size of the central MF bundles. At 48 h after wounding, strongly stained central MF bundles could be detected in EC up to 0.75 mm from the wound edge. Adjacent to the wounds that had failed to reendothelialize 10 months after denudation, some regions had EC with prominent peripheral MF bundles and others, EC with prominent central MF bundles. At the very edge of the wound, the EC and their MF bundles were oriented with their long axes parallel to the wound edge and perpendicular to the direction of blood flow. The failure of the wounded vessel wall to become fully reendothelialized may be related to the orientation of EC at the wound edge. These results show that EC migration in situ is accompanied by a dramatic change in the organization of MF in which different stages can be identified. Microfilament bundles in rapidly migrating cells in vivo, 24 and 48 h after wounding, resemble stress fibers seen in EC migrating in vitro and in slowly migrating fibroblasts and epithelial cells.     

Microtubules are involved in transport of macromolecules by vesicles in cultured bovine aortic endothelial cells

The macromolecular transport in bovine aortic endothelial monolayers, cultured in vitro, was studied by fluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy. A fluid-phase endocytic tracer, fluorescein isothiocyanate dextran 70 kD (FITC-dextran 70), was found to be transported into and out of endothelial cells via vesicles arranged as chains stretching between the luminal surface and the cell interior and also from cell interior to the abluminal surface. The endocytic activity was reduced by colchicine, which disrupts microtubules, and increased during treatment with cytochalasin B, which blocks microfilament polymerization. These findings indicate that microtubules are required for fluid-phase endocytosis and that microfilaments hinder this process. © 1993 Wiley-Liss, Inc.