Reorganization Of Actin Microfilaments Research Articles

Neutrophils Require Activation to Express Functional Cell-Surface Complement Receptor Immunoglobulin.

The phagocytosis-promoting complement receptor, Complement Receptor Immunoglobulin (CRIg), is exclusively expressed on macrophages. It has been demonstrated that expression in macrophages could be modulated by inflammatory mediators, including cytokines. This raised the possibility that a major phagocyte, the neutrophil, may also express CRIg following activation with inflammatory mediators. Here we show that resting peripheral blood neutrophil lysates subjected to protein analysis by Western blot revealed a 35 kDa CRIg isoform, consistent with the expression of CRIg mRNA by RT-PCR. By flow cytometry, CRIg was detected intracellularly and in very minor amounts on the cell surface. Interestingly, expression on the cell surface was significantly increased to functional levels after activation with inflammatory mediators/neutrophil activators; N-Formylmethionine-leucyl-phenylalanine, tumor necrosis factor (TNF), Granulocyte-Macrophage Colony stimulating Factor (GM-CSF), bacterial lipopolysaccharide, leukotriene B4 and phorbol myristate acetate. The increase in expression required p38 MAP kinase and protein kinase C activation, as well as intracellular calcium. Neutrophils which were defective in actin microfilament reorganization due to a mutation in ARPC1B or inhibition of its upstream regulator, Rac2 lose their ability to upregulate CRIg expression. Inhibition of another small GTPase, Rab27a, with pharmacological inhibitors prevented the increase in CRIg expression, suggesting a requirement for the actin cytoskeleton and exocytosis. Engagement of CRIg on TNF-primed neutrophils with an anti-CRIg monoclonal antibody increased the release of superoxide and promoted the activation of p38 but not ERK1/ERK2 or JNK MAP kinases. The TNF-induced increase in killing of Staphylococcus aureus was blocked by the anti-CRIg antibody. Adding to the anti-microbial role of CRIg, it was found that GM-CSF priming lead to the release of neutrophil extracellular traps. Interestingly in contrast to the above mediators the anti-inflammatory cytokine IL-10 caused a decrease in basal expression and GM-CSF induced increase in CRIg expression. The data demonstrate that neutrophils also express CRIg which is regulated by inflammatory mediators and cytokines. The findings show that the neutrophil antimicrobial function involving CRIg requires priming as a means of arming the cell strategically with microbial invasion of tissues and the bloodstream.

The symbiotic role of the actin filament cytoskeleton.

This article is a Commentary on Zhang et al., 221: 1049–1059.

Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells.

Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES.

Formin 1 Regulates Ectoplasmic Specialization in the Rat Testis Through Its Actin Nucleation and Bundling Activity.

During spermatogenesis, developing spermatids and preleptotene spermatocytes are transported across the adluminal compartment and the blood-testis barrier (BTB), respectively, so that spermatids line up near the luminal edge to prepare for spermiation, whereas preleptotene spermatocytes enter the adluminal compartment to differentiate into late spermatocytes to prepare for meiosis I/II. These cellular events involve actin microfilament reorganization at the testis-specific, actin-rich Sertoli-spermatid and Sertoli-Sertoli cell junction called apical and basal ectoplasmic specialization (ES). Formin 1, an actin nucleation protein known to promote actin microfilament elongation and bundling, was expressed at the apical ES but limited to stage VII of the epithelial cycle, whereas its expression at the basal ES/BTB stretched from stage III to stage VI, diminished in stage VII, and was undetectable in stage VIII tubules. Using an in vitro model of studying Sertoli cell BTB function by RNA interference and biochemical assays to monitor actin bundling and polymerization activity, a knockdown of formin 1 in Sertoli cells by approximately 70% impeded the tight junction-permeability function. This disruptive effect on the tight junction barrier was mediated by a loss of actin microfilament bundling and actin polymerization capability mediated by changes in the localization of branched actin-inducing protein Arp3 (actin-related protein 3), and actin bundling proteins Eps8 (epidermal growth factor receptor pathway substrate 8) and palladin, thereby disrupting cell adhesion. Formin 1 knockdown in vivo was found to impede spermatid adhesion, transport, and polarity, causing defects in spermiation in which elongated spermatids remained embedded into the epithelium in stage IX tubules, mediated by changes in the spatiotemporal expression of Arp3, Eps8, and palladin. In summary, formin 1 is a regulator of ES dynamics.

RpS6 regulates blood-testis barrier dynamics through Arp3-mediated actin microfilament organization in rat sertoli cells. An in vitro study.

In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.

Effect of Cancer Drug LHRH-PE40 on the Elasticity of Living HeLa Cells

The quantitative analysis of biomechanical profiles at the single-cell level can provide additional information. The careful analysis of biomechanical properties at the single-cell level can provide additional information that is usually not available using traditional biological methods, but may be crucial to assess and understand tumor progress and response to chemotherapy. In this study, the on-line changes of cell elasticity during the exposure to immunotoxin LHRH-PE40 were monitored by atomic force microscopy (AFM) on living HeLa cell surface under physiological condition. The results from AFM based force spectroscopy showed that LHRH-PE40 induced a distinct increase of the cell surface stiffness of HeLa cells. The fluorescence images implied that the cancer drug LHRH-PE40 would affect the re-organization of actin microfilaments, which led to the increase of the stiffness of HeLa cells.

Isolation of tropomyosin particles from cultured cell cytosol and their protein composition assay

The presence of an actin-binding protein, tropomyosin, in particles or protein complexes not bound with actin structures were found during an assay of structural rearrangements of actin cytoskeleton. To study the composition and properties of these protein complexes, a novel method of their isolation without destroying cytoskeleton structures has been elaborated. The protein composition of isolated tropomyosin particles was assessed by gel filtration, electrophoresis, and Western blotting. It was demonstrated that they are about 700-kDa multimolecular complexes. In addition to tropomyosin and actin, these complexes contained Hsp70, Hsp90, and myosin-9 identified by mass spectrometry. It was found that the deacetylase inhibitor, trichostatin A, which induced actin cytoskeleton rearrangements, changed the number of tropomyosin particles and caused redistribution of tropomyosin between cytosol and cytoskeleton. These results demonstrate that these multimolecular complexes may participate in the process of reorganization of actin microfilaments.

Elicitor-Induced Cytoskeletal Rearrangement Relates to Vacuolar Dynamics and Execution of Cell Death: In Vivo Imaging of Hypersensitive Cell Death in Tobacco BY-2 Cells

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.

Noise Yields Order - Auxin, Actin, and Polar Patterning

Plant patterns have to integrate environmental cues and to cope with a high level of noise in the sensory outputs of individual cells. In the first part of this review, we demonstrate that local self-amplification linked to lateral inhibition can meet this requirement. In the second part, we describe the search for candidates for such self-amplification loops in the context of auxin-dependent cell growth using Graminean coleoptiles as a model. Auxin-dependent reorganization of actin microfilaments interfered with the auxin sensitivity of growth. Auxin might control the intracellular transport of factors important for auxin sensing via the actomyosin system. By means of a rice mutant with elevated auxin responsiveness, we identified an auxin response factor (OSARF1), whose expression is upregulated by auxin as a second candidate for a self-amplification loop. We studied the cross-talk between auxin signalling and environmental cues in the rice mutant hebiba, where the photoinhibition of growth is impaired. We found that jasmonate plays a central role in this cross-talk correlated to a downregulation of auxin responsiveness. To obtain an insight into auxin-dependent coordination, we analyzed a tobacco cell line with axial cell divisions. By a combination of modelling and physiological manipulation, we could demonstrate that auxin synchronizes the divisions of adjacent cells on the background of strong heterogeneity of individual cells. We conclude that self-amplification of auxin signalling coupled to mutual competition for available auxin provides a versatile tool to fulfill the special requirements posed by patterning in plants.

Nonhost Resistance in Arabidopsis-Colletotrichum Interactions Acts at the Cell Periphery and Requires Actin Filament Function

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.

Microtubules regulate aortic endothelial cell actin microfilament reorganization in intact and repairing monolayers.

To understand the role of microtubules and microfilaments in regulating endothelial monolayer integrity and repair, and since microtubules and microfilaments show some co-alignment in endothelial cells, we tested the hypothesis that microtubules organize microfilament distribution. Disruption of microtubules with colchicine in resting confluent aortic endothelial monolayers resulted in disruption of microfilament distribution with a loss of dense peripheral bands, an increase in actin microfilament bundles, and an associated increase of focal adhesion proteins at the periphery of the cells. However, when microfilaments were disrupted with cytochalasin B, microtubule distribution did not change. During the early stages of wound repair of aortic endothelial monolayers, microtubules and microfilaments undergo a sequential series of changes in distribution prior to cell migration. They are initially distributed randomly relative to the wound edge, then align parallel to the wound edge and then elongate perpendicular to the wound edge. When microtubules in wounded cultures were disrupted, dense peripheral bands and lamellipodia formation were lost with increases in central stress fibers. However, following microfilament disruption, microtubule redistribution was not disrupted and the microtubules elongated perpendicular to the wound edge similar to non-treated cultures. Microtubules may organize independently of microfilaments while microfilaments require microtubules to maintain normal organization in confluent and repairing aortic endothelial monolayers.

The Metastasis Suppressor Gene KiSS-1 Encodes Kisspeptins, the Natural Ligands of the Orphan G Protein-coupled Receptor GPR54

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.

Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation.

T-cell activation requires clustering of a threshold number of T-cell receptors (TCRs) at the site of antigen presentation, a number that is reduced by CD28 co-receptor recruitment of signalling proteins to TCRs. Here we demonstrate that a deficiency in beta1,6 N-acetylglucosaminyltransferase V (Mgat5), an enzyme in the N-glycosylation pathway, lowers T-cell activation thresholds by directly enhancing TCR clustering. Mgat5-deficient mice showed kidney autoimmune disease, enhanced delayed-type hypersensitivity, and increased susceptibility to experimental autoimmune encephalomyelitis. Recruitment of TCRs to agonist-coated beads, TCR signalling, actin microfilament re-organization, and agonist-induced proliferation were all enhanced in Mgat5-/- T cells. Mgat5 initiates GlcNAc beta1,6 branching on N-glycans, thereby increasing N-acetyllactosamine, the ligand for galectins, which are proteins known to modulate T-cell proliferation and apoptosis. Indeed, galectin-3 was associated with the TCR complex at the cell surface, an interaction dependent on Mgat5. Pre-treatment of wild-type T cells with lactose to compete for galectin binding produced a phenocopy of Mgat5-/- TCR clustering. These data indicate that a galectin-glycoprotein lattice strengthened by Mgat5-modified glycans restricts TCR recruitment to the site of antigen presentation. Dysregulation of Mgat5 in humans may increase susceptibility to autoimmune diseases, such as multiple sclerosis.

Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes.

We examined the temporal reorganization of actin microfilaments by insulin and its participation in the localization of signaling molecules and glucose transporters in L6 myotubes expressing myc-tagged glucose transporter 4 (GLUT4myc). Scanning electron microscopy revealed a dynamic distortion of the dorsal cell surface (membrane ruffles) upon insulin treatment. In unstimulated cells, phalloidin-labeled actin filaments ran parallel to the longitudinal axis of the cell. Immunostaining of the p85 regulatory subunit of phosphatidylinositol 3-kinase was diffusely punctate, and GLUT4myc was perinuclear. After 3 minutes of insulin treatment, actin reorganized to form structures; these structures protruded from the dorsal surface of the myotubes by 10 minutes and condensed in the myoplasm into less prominent foci at 30 minutes. The p85 polypeptide colocalized with these structures at all time points. Actin remodeling and p85 relocalization to actin structures were prevented by cytochalasin D or latrunculin B. GLUT4myc recruitment into the actin-rich projections was also observed, but only after 10 minutes of insulin treatment. Irrespective of insulin stimulation, the majority of p85 and a portion (45%) of GLUT4 were recovered in the Triton X-100-insoluble material that was also enriched with actin. In contrast, vp165, a transmembrane aminopeptidase that morphologically colocalized with GLUT4 vesicles, was fully soluble in Triton X-100 extracts of both insulin-treated and control myotubes. Transient transfection of dominant inhibitory Rac1 (N17) into L6 myotubes prevented formation of dorsal actin structures and blocked insulin-induced GLUT4myc translocation to the cell surface. We propose that insulin-dependent formation of actin structures facilitates the association of PI3-K (p85) with GLUT4 vesicles and, potentially, the arrival of GLUT4 at the cell surface.

The cytotoxic necrotizing factor 1 (CNF1) from uropathogenic Escherichia coli.

Escherichia coli are an important cause of intestinal infections. These bacteria, become highly pathogenic upon acquision of genes coding for virulence factors. In the case of urinary tract, infections (UTIs), E. coli strains often express exotoxins, such as hemolysin and cytotoxic necrotizing factor 1 (CNF1) together with colonization factors such as Pap or Afa . Genes coding for these virulence factors are linked on the chromosome of uropathogenic E. coli J96 strains clustered within chromosomal DNA which have been called pathogenicity islands PAIs). CNF1 is a toxin of 110 kDa which induces, in cultured cells, a reorganization of actin microfilaments by activation of the 21-kDa Rho GTP-binding protein. adhesins

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.

Early alterations of actin cytoskeleton in OK cells by opioids.

Recently we identified and characterized opioid binding sites in OK (opossum kidney) cells and observed decreased proliferation of these cells in response to opioids. In the present study we investigated the effects of opioids on the actin cytoskeleton and explored whether their antiproliferative action may relate to alterations in the distribution or the dynamics of actin microfilaments. Exposure of OK cells to the opioids alphaS1 casomorphin and ethylketocyclazocine resulted in a rapid and substantial actin microfilament reorganization. This was documented by a significant dose-dependent decrease in the amounts of F-actin, determined by measurements of quantitative fluorescence, by immunoblot analysis and by a concomitant increase of the G/total-actin ratio measured by the DNase I inhibition assay. These changes were verified by confocal laser scanning microscopy, which showed marked redistribution of the microfilamentous structures in the presence of the opioids without affecting the organization of microtubules or vimentin intermediate filaments. The effect of opioids on actin polymerization dynamics occurred within 15 min and persisted for at least 2 h, while their restoration to control levels was accomplished 6 h later, indicating a reversible phenomenon. Northern blot analysis showed that the concentration of the actin transcript was unaffected. The addition of diprenorphine, a general opioid antagonist, prevented the effects of opioids on the actin cytoskeleton. The inhibition of OK cell proliferation, induced by ethylketocyclazocine and alphaS1 casomorphin was partially prevented in the presence of phallacidin, which stabilizes microfilaments. Our findings demonstrate that opioids, acting via kappa 1 binding sites, induce rapidly modifications in the dynamics of actin polymerization, and in the organization of microfilaments in OK cells, which may relate to their antiproliferative effect on these cells.

Thrombospondin Signaling of Focal Adhesion Disassembly Requires Activation of Phosphoinositide 3-Kinase

Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17-35. Use of a synthetic peptide (hep I), containing amino acids 17-35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of alpha-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2-3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In 32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.

Reorganization of actin microfilaments and microtubules in regenerating retinal pigment epithelium

Retinal pigment epithelium (RPE) regenerating after experimental damage in rabbits exhibits major changes in cell shape, polarity and junctions--features that depend on the cytoskeleton. This report correlates these changes with the redistribution of actin microfilaments and microtubules, using electron microscopy and confocal laser scanning microscopy. We compare immature cells with the more mature cells that form the new epithelial monolayer. Two populations of immature RPE cells are interspersed at the edge of the regenerating RPE sheet. One population of immature cells makes few junctions with their neighbors or the basement membrane. They form pseudopodia and exhibit a prominent network of actin microfilaments beneath the plasma membrane. These cells are probably motile and advance the epithelial sheet. Another population of immature cells contains numerous stress fibers that insert into large basement membrane attachments. The cells make focal adhesions with their neighbors, rather than the junctional complexes characteristic of mature RPE cells. These cells are probably not motile and mature into the cells forming the new monolayer--cuboidal cells with numerous basal folds and apical villi and a complete belt of intercellular junctions. Stress fibers are lost as the circumferential bundle associated with the zonula adherens re-forms. Microtubules, which form prominent longitudinal bundles running through the processes of immature cells, take on the meshwork organization characteristic of mature RPE as the immature cells differentiate.