Focal adhesion proteins, vinculin and integrin ß5, during early pregnancy in rat uterine epithelial cells: anastrozole favors their normal distribution / Proteínas de adhesión focales, vinculina e integrina ß5, durante el embarazo temprano en células epiteliales uterinas de rata: anastrozol favorece su distribución normal

SUMMARY: An alternative superovulator to replace clomiphene citrate is needed as clomiphene citrate is associated with low pregnancy rates. Anastrozole is an effective superovulator, but it has not been well researched. In order to determine the effectiveness of anastrozole as a superovulator and to compare it with clomiphene citrate in similar situations, this study ascertained the effects of these drugs on the expression of the focal adhesion proteins, vinculin and integrin β5, which are uterine receptivity markers, in the uterine epithelial cells of day 1 and day 6 pregnant Wistar rats. The results show that vinculin and integrin β5 are co-localized at the base of the uterine epithelium at day 1 of pregnancy whereas at day 6, they disassemble from the basal focal adhesions and co-localize and significantly increase their expression apically (p≤0.0001). Moreover, there is a significant difference in the protein expression levels of vinculin and integrin b5 in uterine luminal epithelial cells between untreated (control) and chlomiphene citrate treated rats (p≤0.0001), anastrozole and chlomiphene citrate treated rats at day 6 (p≤0.0001) suggesting the interpretation that anastrozole seems to enhance their expression in order to perhaps assist in the implantation process of the blastocyst. The immunofluorescence experiments agree with the vinculin and integrin β5 gene expression findings in which at day 6 of pregnancy, vinculin and integrin β5 gene expression are significantly upregulated in uterine luminal epithelial cells in the anastrozole treated group relative to the calibrator sample (p≤0.0001). These findings suggest that anastrozole is implantation friendly.

RESUMEN: Es necesario un superovulador alternativo para reemplazar el citrato de clomifeno, debido a que está asociado con bajas tasas de preñez. El anastrozol es un superovulador eficaz, sin embargo es poca su investigación. Con el fin de determinar la efectividad del anastrozol como superovulador y compararlo con citrato de clomifeno en situaciones similares, se determinaron los efectos de estos fármacos sobre la expresión de las proteínas de adhesión focal, vinculina e integrina β5, en marcadores de receptividad uterina en días 1 y 6, en las células epiteliales uterinas de ratas Wistar preñadas. Los resultados muestran que la vinculina y la integrina β5 se co-localizan en la base del epitelio uterino al día 1 de la gravidez mientras que al día 6 se desmontan de las adherencias focales basales, co-localizan y aumentan significativamente su expresión apicalmente (p≤0.0001). Además, existe una diferencia significativa en los niveles de expresión de proteína de vinculina e integrina β5 en células epiteliales luminales uterinas entre ratas no tratadas (control) y tratadas con citrato declomifeno (p≤0.0001), ratas tratadas con anastrozol y citrato declomifeno al día 6 (p≤0,0001) sugiriendo la interpretación de que el anastrozol parece mejorar su expresión con el fin de ayudar en el proceso de implantación del blastocisto. Los experimentos de inmunofluorescencia coinciden con los resultados de la expresión de los genes vinculina e integrina β5 en los cuales al día 6 de la preñez, la vinculina y la integrina β5 están significativamente reguladas en células epiteliales luminales uterinas en el grupo tratado con anastrozol con respecto a la muestra del calibrador (p<0,0001). Estos hallazgos sugieren que el anastrozol es favorable para la implantación.

Impaired cytoskeletal arrangements and failure of ventral body wall closure in chick embryos treated with rock inhibitor (Y-27632).

AIM: Rho-associated kinase (ROCK) signaling regulates numerous fundamental developmental processes during embryogenesis, primarily by controlling actin-cytoskeleton assembly and cell contractility. ROCK knockout mice exhibit a ventral body wall defect (VBWD) phenotype due to disorganization of actin filaments at the umbilical ring. However, the exact molecular mechanisms leading to VBWD still remain unclear. Improper somitogenesis has been hypothesized to contribute to failure of VBW closure. We designed this study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) disrupts cytoskeletal arrangements in morphology during early chick embryogenesis, which may contribute to the development of VBWD. METHODS: At 60 h incubation, chick embryos were explanted into shell-less culture and treated with 50 µL of vehicle for controls (n = 33) or 50 µL of 500 µM of Y-27632 for the experimental group (Y-27, n = 56). At 8 h post-treatment, RT-PCR was performed to evaluate mRNA levels of N-cadherin, E-cadherin and connexin43. Immunofluorescence confocal microscopy was performed to analyze the expression and distribution of actin, vinculin and microtubules in the neural tube and somites. A further cohort of embryos was treated in ovo by dropping 50 µL of vehicle or 50 µL of different concentrations of Y-27632 onto the embryo and allowing development to 12 and 14 days for further assessment. RESULTS: Gene expression levels of N-cadherin, E-cadherin and connexin43 were significantly decreased in treated embryos compared with controls (p < 0.05). Thickened actin filament bundles were recorded in the neural tube of Y-27 embryos. In somites, cells were dissociated with reduced actin distribution in affected embryos. Clumping of vinculin expression was found in the neural tube and somites, whereas reduced expression of microtubules was observed in Y-27 embryos compared with controls. At 12 and 14 days of development, affected embryos presented with an enlarged umbilical ring and herniation of abdominal contents through the defect. CONCLUSION: ROCK inhibition alters cytoskeletal arrangement during early chick embryogenesis, which may contribute to failure of anterior body wall closure causing VBWD at later stages of development.

Promotion of Dental Pulp Cell Migration and Pulp Repair by a Bioceramic Putty Involving FGFR-mediated Signaling Pathways.

Mineral trioxide aggregate is the currently recommended material of choice for clinical pulp repair despite several disadvantages, including handling inconvenience. Little is known about the signaling mechanisms involved in bioceramic-mediated dental pulp repair-particularly, dental pulp cell (DPC) migration. This study evaluated the effects of iRoot BP Plus, a novel ready-to-use nanoparticulate bioceramic putty, on DPC migration in vitro and pulp repair in vivo, focusing on possible involvement of fibroblast growth factor receptor (FGFR)-related signaling, including mitogen-activated protein kinase and Akt pathways. Treatment with iRoot BP Plus extracts enhanced horizontal and vertical migration of DPCs, which was comparable with the effects induced by mineral trioxide aggregate extracts. The DPCs exposed to iRoot BP Plus extracts demonstrated no evident apoptosis. Importantly, treatment with iRoot BP Plus extracts resulted in rapid activation of FGFR, p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), and Akt signaling in DPCs. Confocal immunofluorescence staining revealed that iRoot BP Plus stimulated focal adhesion formation and stress fiber assembly in DPCs, in addition to upregulating the expression of focal adhesion molecules, including p-focal adhesion kinase, p-paxillin, and vinculin. Moreover, activation of FGFR, ERK, JNK, and Akt were found to mediate the upregulated expression of focal adhesion molecules, stress fiber assembly, and enhanced DPC migration induced by iRoot BP Plus. Consistent with the in vitro results, we observed induction of homogeneous dentin bridge formation and expression of p-focal adhesion kinase, p-FGFR, p-ERK 1/2, p-JNK, and p-Akt near injury sites by iRoot BP Plus in an in vivo pulp repair model. These data demonstrate that iRoot BP Plus can promote DPC migration and pulp repair involving the FGFR-mediated ERK 1/2, JNK, and Akt pathways. These findings provide valuable insights into the signaling mechanisms underlying nanoparticulate bioceramic-mediated pulp repair.

Simvastatin disrupts cytoskeleton and decreases cardiac fibroblast adhesion, migration and viability.

Statins reduce the isoprenoids farnesyl and geranylgeranyl pyrophosphate, essential intermediates, which control a diversity of cellular events such as cytoskeleton integrity, adhesion, migration and viability. Cardiac fibroblasts are the major non-myocyte cell constituent in the normal heart, and play a key role in the maintenance of extracellular matrix. The effects of simvastatin on cardiac fibroblast processes previously mentioned remain unknown. Our aims were to investigate the effects of simvastatin on cytoskeleton structure and focal adhesion complex assembly and their relationships with cell adhesion, migration and viability in cultured cardiac fibroblasts. To this end, cells were treated with simvastatin for 24 h and changes in actin cytoskeleton, levels of vimentin and paxillin as well as their subcellular localization were analyzed by Western blot and immunocytochemistry, respectively. Cell adhesion to plastic or collagen coated dishes, migration in Transwell chambers, and cell viability were analyzed after simvastatin treatment. Our results show that simvastatin disrupts actin cytoskeleton and focal adhesion complex evaluated by phalloidin stain and immunocytochemistry for paxillin and vinculin. All these effects occurred by a cholesterol synthesis-independent mechanism. Simvastatin decreased cell adhesion, migration and viability in a concentration-dependent manner. Finally, simvastatin decreased angiotensin II-induced phospho-paxillin levels and cell adhesion. We concluded that simvastatin disrupts cytoskeleton integrity and focal adhesion complex assembly in cultured cardiac fibroblasts by a cholesterol-independent mechanism and consequently decreases cell migration, adhesion and viability.

Down-regulation of vinculin upon MK886-induced apoptosis in LN18 glioblastoma cells.

Glioblastomas are a type of malignant brain tumor and are among the most difficult cancers to treat. One strategy to treat aggressive cancers is the use of drugs that target multiple signaling pathways. MK886 is a drug known to inhibit both 5- lipoxygenase-activating-protein (FLAP) and peroxisome proliferator activated receptor-alpha (PPAR-alpha). The objectives of this study were to investigate the ability of MK886 to induce apoptotic cell death in LN18 glioblastoma cells and to characterize the cell death mechanisms. MK886 induced massive apoptotic LN18 cell death that was manifested by the release of nucleosomes, annexinV binding to phosphatidylserine in the absence of nuclear staining, and changes in the fluorescent intensity of Mito Tracker Deep Red 633 indicating changes in mitochondrial oxidative function and mass. The alteration of the mitochondrial function implied that MK886 induced apoptosis in LN18 cells via a mitochondrial pathway. The broad caspases inhibitor ZVAD-FMK inhibited MK886-induced nucleosome release, but not annexinV binding or MK886-altered mitochondrial function. Real time RT-PCR demonstrated that LN18 cells expressed significant levels of FLAP and PPAR- alpha mRNAs. A low level of arachidonate 5-lipoxygenase (ALOX-5) mRNA was detected, but little, if any, arachidonate 12- lipoxygenase (ALOX-12) mRNA was present. In addition, MK886-induced apoptosis in LN18 cells was accompanied by a decrease in the protein and mRNA levels of vinculin, but not other focal adhesion proteins. In summary, the data presented here indicate that disruption of the actin-vinculin-cell-cytoskeleton matrix of the LN18 glioblastoma is a component of the MK886 induced apoptosis. In addition, MK886 treated LN18 cells could provide one model in which to investigate drugs that target lipoxygenase and PPAR-alpha pathways in the chemotherapeutic treatment of glioblastomas.

The lipoxygenase inhibitor, baicalein, modulates cell adhesion and migration by up-regulation of integrins and vinculin in rat heart endothelial cells.

BACKGROUND AND PURPOSE: Endothelial cell proliferation, migration and adhesion are necessary for the formation of new blood vessels. We reported previously that baicalein strongly inhibited proliferation of rat heart endothelial cells and here we assess effects on migration and adhesion of these cells. EXPERIMENTAL APPROACH: Effects of baicalein on endothelial migration and adhesion were determined by in vitro wound assays and in modified Boyden chambers. Protein expression and subcellular distribution in rat heart endothelial cells were analysed by immunoblots and immunofluorescence staining. RESULTS: Pretreatment with baicalein for 48 h resulted in a concentration-dependent inhibition of endothelial migration, with an IC(50) of approximately 20 microM. Adhesion assays revealed that baicalein stimulated endothelial cell adhesion to fibronectin and vitronectin, effects blocked by the synthetic peptide Arg-Gly-Asp (RGD). Moreover, treatment with a blocking antibody against integrin alpha5beta1 drastically attenuated baicalein-mediated endothelial adhesion to fibronectin, but not to vitronectin. Furthermore, baicalein-mediated anti-migration effect and adhesion promotion could be partially reversed by the addition of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Western blot analysis indicated that baicalein increased expression levels of integrin-alpha5beta1, -alphavbeta3 and vinculin proteins. Immunofluorescence staining showed that baicalein induced a marked reorganization of actin stress fibres and the recruitment of vinculin and integrins to focal adhesion plaques, with consequently increased formation of focal adhesion contacts. CONCLUSIONS AND IMPLICATIONS: Baicalein markedly inhibited the migration and enhanced the adhesion of rat heart endothelial cells, possibly by up-regulation of the integrins (alpha5beta1 and alphavbeta3) and vinculin and by promotion of actin reorganization and focal adhesion contact formation.

Effects of two-step freezing on the ultra-structural components of murine osteoblast cultures.

Understanding the ultra-structural response of cells to the cryopreservation process is important for designing cryopreservation strategies for cells and tissues. Cell-cell interaction and cell-scaffold interactions alter cryopreservation response and, in turn, the cellular structures involved in adhesion and intercellular contact are possible targets of cryopreservation-induced damage. Immuno-fluorescence was used to assess the status of the actin filaments (F-actin), focal adhesions (vinculin) and gap junctions (connexin-43) of murine osteoblasts attached to hydroxyapatite (HA) discs and plastic coverslips for a two-step freezing process. The freezing process de-polymerized and distorted the actin filaments of dead cells, while those of live cells experienced little change. Vinculin and connexin-43 structures were rarely seen in dead cells, while a portion of vinculin (8.14+/-2.27 percent) and connexin-43 (21.7+/-4.7 percent) structures remained in live cells. These results suggest that focal adhesions and gap junctions may support cell robustness during cryopreservation. The present study contributes to our knowledge of the damage mechanisms associated with attached cells during a freezing process.

Inhibition of adipogenesis by RGD-dependent disintegrin.

Adipogenesis plays a central role in obesity development. The processes of adipogenesis include migration, adhesion, proliferation and survival of preadipocytes and differentiation to mature adipocytes. Many of these biological functions are related to integrins. Here, we found that snake venom-derived arginine-glycine-aspartic acid (RGD)-containing disintegrin inhibited adipogenesis. Rhodostomin but not rhodostomin RGD mutants (RGE-Rn and AKGDWN-Rn) caused the detachment of primary cultured preadipocyte. Furthermore, rhodostomin also inhibited focal adhesion of preadipocyte, including the inhibition of the expression of focal adhesion kinase (FAK) and FAK phosphorylation, assembly of vinculin and reorganization of actin cytoskeleton. Cell viability of preadipocytes was decreased after rhodostomin treatment in a concentration-dependent manner. The results of flow cytometric analysis showed that rhodostomin induced cell apoptosis. In addition, chromatin condensation was observed in DAPI staining. The increase of Bax expression and activation of capsase-3 was detected following rhodostomin treatment. Addition of dexamethasone, IBMX and insulin induced differentiation of preadipocytes into mature adipocytes and treatment of cells with rhodostomin during the initial 3 days showed less mature adipocytes following 9-10 days of differentiating period. The triglyceride content and gene expression of peroxisome proliferators-activated receptor gamma (PPARgamma) and leptin also decreased in response to the treatment of rhodostomin. These results suggest that disintegrin inhibits processes of adipogenesis and may be developed to treat obesity.

Nonspecific depolarization of the plasma membrane potential induces cytoskeletal modifications of bovine corneal endothelial cells in culture.

Modifications in the cell membrane potential have been suggested to affect signaling mechanisms participating in diverse cellular processes, many of which involve structural cellular alterations. In order to contribute some evidence in this respect, we explored the effects of several depolarizing procedures on the structure and monolayer organization of bovine corneal endothelial cells in culture. Visually confluent cell monolayers were incubated with or without the depolarizing agent, either in a saline solution or in culture medium for up to 30 min. Membrane potential was monitored by fluorescence microscopy using oxonol V. Fluorescent probes were employed for F-actin, microtubules, and vinculin. Depolarization of the plasma membrane, achieved via the incorporation of gramicidin D into confluent endothelial cells or by modifications of the extracellular saline composition, provoked an increment of oxonol fluorescence and changes in cell morphology, consisting mainly of modifications in the cytoskeletal organization. In some areas, noticeable intercellular spaces appear. The cytoskeleton modifications mainly consist of a marked redistribution of F-actin and microtubules, with accompanying changes in vinculin localization. The results suggest that the depolarization of the plasma membrane potential may participate in mechanisms involved in cytoskeleton organization and monolayer continuity in corneal endothelial cells in culture.

Gastrin mediated cholecystokinin-2 receptor activation induces loss of cell adhesion and scattering in epithelial MDCK cells.

The presence of gastrin and CCK-2/gastrin receptors in human preneoplastic and neoplastic lesions of pancreas and colon suggests a role in cancer development. Gastrin's growth-promoting action has been established, but a role in cellular morphogenetic processes promoting tumor invasion has been elusive. Our aim was (i) to investigate whether activation of the CCK-2R affects cellular morphology, intercellular adhesion and motility, as crucial parameters of epithelial differentiation, and (ii) to identify the signaling pathways and mechanisms implicated. Madin-Darby Canine Kidney (MDCK) cells were chosen to generate an epithelial non-tumorigenic model system expressing human CCK-2R. Epithelial differentiation and motility were analysed upon CCK-2R activation using immunocytochemistry and invasion assays. The functionality of adhesion complexes and activity of signaling proteins was determined with biochemical techniques. CCK-2R activation induced cell dissociation and enhanced invasion, preceded by decreased membrane localization of adherens junction molecules and nuclear accumulation of beta-catenin. Concomitantly, and requiring the activation of several signaling pathways, catenins were shifted from the cytoskeletal to the cytoplasmic fraction, suggesting the detachment of the cytoskeleton from the adherens complex. These data represent the first evidence for the CCK-2R, regulating cell-cell and cell-substrate adhesion and support a role for CCK-2R in the progression of carcinoma.

The effect of 12-O-tetradecanoylphorbol-13-acetate on Sertoli cell morphology and cytoskeletal organization: an in vitro study by means of cryo-SEM and fluorescent techniques.

By means of cryo-scanning electron microscopy (cryo-SEM) and fluorescent techniques, evidence is provided on how 12-O-tetradecanoylphorbol-13-acetate (TPA) affects Sertoli cell morphology and F-actin and vinculin organization in vitro. In order to visualize the morphological changes, the cells were observed with cryo-SEM. F-actin was localized using rhodamine (TRI)-phalloidin and vinculin using a primary monoclonal antibody and a second TRI-conjugated antibody. The results indicate that after the addition of 10(-7) M TPA, Sertoli cells begin to round up and their cytoplasm is retracted towards a central region. Actin bundle organization is disrupted and vinculin assumes a punctuate distribution throughout the cell. Thus, the reorganization of actin and vinculin and subsequent changes in cell morphology seem to be brought about by TPA affecting not only actin but also the protein vinculin which interacts with actin. A discussion is made concerning the effect of TPA on cytoskeletal reorganization, which is closely related to cell transformation.

Organization of focal adhesion plaques is disrupted by action of the HIV-1 protease.

Focal adhesion plaques were severely affected in human embryonic fibroblasts permeabilized with digitonin and incubated in buffer containing the human immunodeficiency virus type 1 protease (HIV-1 PR). A mutant HIV-1 PR (3271 HIV-1 PR) had no effect on focal adhesion plaques. Similar effects were seen with cells microinjected with either HIV-1 PR or 3271 HIV-1 PR. Immunoblots of the human embryonic fibroblasts demonstrated that a number of focal adhesion plaque proteins were specifically cleaved by HIV-1 PR. These included fimbrin, focal adhesion plaque kinase (FAK), talin, and, to a lesser extent, filamin, spectrin and fibronectin. Proteins detected by antibodies to beta 4 integrin and alpha 3 integrin were also cleaved by the HIV-1 PR. Control experiments demonstrated that the effect and protein cleavages described are due to action of the HIV-1 PR and not to the action of endogenous host cell proteases.

Src-/- fibroblasts are defective in their ability to disassemble focal adhesions in response to phorbol ester/hyaluronan treatment.

Exogenous hyaluronan promotes a rapid recruitment of Src to lamellae of mutant active H-ras transformed fibroblasts and an Src- and RHAMM (CD168)-dependent increase in random motility. These responses are accompanied by a loss of vinculin-positive lamellae focal adhesions. Nontransformed immortalized wild-type fibroblasts (WT) do not increase random motility in response to hyaluronan alone, but do increase motility in response to a combination of PMA treatment followed by hyaluronan. PMA treatment alone increases the number of lamellae/cell, percentage of cells with lamellae and number of focal adhesions/lamellae. Subsequent addition of hyaluronan does not affect the number of lamellae/cell but reduces both the number of focal adhesion/lamellae and the percentage of cells forming focal adhesion-positive lamellae. These effects are prevented by blocking RHAMM antibodies and mimicked by agonist RHAMM antibodies. Src-/- fibroblasts exhibit a limited response to PMA but do not increase motility or disassemble focal adhesions in response to a subsequent addition of HA. Rescue of Src-/- fibroblasts with either SrcA or c-Src restores response to close to WT levels. These results suggest that Src activity is uniquely required for both PMA and PMA-induced hyaluronan regulation of random motility and focal adhesion turnover.

Rho family GTPases regulate VEGF-stimulated endothelial cell motility.

Migration of endothelial cells induced by vascular endothelial growth factor (VEGF) is a critical step in angiogenesis. Stimulation of motility by growth factors such as VEGF requires interaction with the signal transduction pathways activated by the extracellular matrix (ECM). Here we demonstrate that the Rac GTPase is the critical intersection activated by type 1 collagen ECM and VEGF during stimulation of endothelial cell motility. To analyze the role of the Rho family GTPases in VEGF-stimulated endothelial cell chemotaxis and ECM-stimulated haptotaxis, we transduced the respective fusion proteins in human foreskin dermal endothelial cells using a Tat peptide from the human immunodeficiency virus Tat protein. VEGF signaling required Rac activation during chemotaxis, and Rac and Cdc42 were activated during haptotaxis on type I collagen. Similar to VEGF, Rac activation induced an increase in endothelial cell stress fiber and focal adhesion. Surprisingly, Rho activation was not present in collagen-induced haptotaxis or stimulation of chemotaxis by VEGF, although Rho induced stress fibers and focal adhesions similar to Rac activation. The result of constitutive Rho activation was an inhibition of haptotaxis. Thus, Rac is required and sufficient for the activation of endothelial cell haptotaxis and VEGF-stimulated chemotaxis.

Actin activates a cryptic dimerization potential of the vinculin tail domain.

The tail domain of vinculin (V(t)) is an actin binding module containing two regions that interact with F-actin. Although intact V(t) purified from a bacterial expression system is a globular monomer, each actin binding region dimerizes when expressed individually, suggesting the presence of cryptic self-association sites whose exposure is regulated. We show that actin modulates V(t) self-association by inducing or stabilizing a conformational change in V(t) that allows dimerization. Chemical cross-linking studies implicate one of the actin binding regions in mediating dimerization in the presence of actin. Actin-induced V(t) dimers may play a role in the filament cross-linking activity of this protein. The V(t) dimers induced by actin are biochemically distinct from the V(t) dimers and higher oligomers induced by acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate, suggesting structural differences in V(t) bound to these two ligands that may provide a mechanistic basis for inhibition of F-actin binding by phosphatidylinositol 4,5-bisphosphate. The ability of actin to regulate the dimerization state of an actin binding protein suggests that, rather than serving a passive structural role, actin filaments may directly participate in signal transduction and other cellular events that are known to depend on cytoskeletal integrity.

Bacterial lipopolysaccharide induces cytoskeletal rearrangement in small intestinal lamina propria fibroblasts: actin assembly is essential for lipopolysaccharide signaling.

Cytoskeletal proteins are major components of the cell backbone and regulate cell shape and function. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) on the dynamics and organization of the cytoskeletal proteins, actin, vimentin, tubulin and vinculin in human small intestinal lamina propria fibroblasts (HSILPF). A noticeable change in the actin architecture was observed after 30 min incubation with LPS with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 2 h. Reorganization of the vimentin network into vimentin bundling was conspicuous at 2 h. With further increase in the time period of LPS exposure, diffused staining of vimentin along with vimentin bundling was observed. Vinculin plaques distributed in the cell body and cell periphery in the control cells rearrange to cell periphery in LPS-treated cells by 30 min of LPS exposure. However, there was no change in the tubulin architecture in HSILPF in response to LPS. LPS increased the F-actin pool in HSILPF in a concentration-dependent manner with no difference in the level of G-actin. A time-dependent study depicted an increase in the G-actin pool at 10 and 20 min of LPS exposure followed by a decrease at further time intervals. The F-actin pool in LPS-treated cells was lower than the control levels at 10 and 20 min of LPS exposure followed by a sharp increase until 120 min and finally returning to the basal level at 140 and 160 min. Further (35)S-methionine incorporation studies suggested a new pool of actin synthesis, whereas the synthesis of other cytoskeletal filaments was not altered. Cytochalasin B, an actin-disrupting agent, severely affected the LPS induced increased percentage of 'S' phase cells and IL-6 synthesis in HSILPF. We conclude that dynamic and orchestrated organization of the cytoskeletal filaments and actin assembly in response to LPS may be a prime requirement for the LPS induced increase in percentage of 'S' phase cells and IL-6 synthesis

Interrelation of motility, cytoskeletal organization and gap junctional communication with invasiveness of melanocytic cells in vitro.

Intercellular communication and the active movement of malignant cells into and through host tissue barriers play a critical role during the complex process of tumor invasion. Motile activity, cytoskeletal actin and vinculin organization as well as gap junctional communication of in vivo benign and malignant melanocytes were compared and related to in vitro invasiveness. Normal melanocytes, Melan-a, showed significantly less motile activity, a higher organization of the actin cytoskeleton and more vinculin-containing cell-substratum adhesion plaques than highly metastatic melanoma cells, K1735-M2. There was no pronounced difference in gap junctional communication under comparable culture conditions. However, cultivation of Melan-a cells in a conventional melanocyte growth medium containing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced intercellular communication. Melanocytes were less invasive than melanoma cells both in the embryonic chick heart model and in the Matrigel invasion assay. The least invasive activity was determined for melanocytes cultivated in TPA-deficient medium indicating that the medium supplement TPA stimulates invasion. The comparison of certain in vitro properties of both melanocytic cell lines revealed a positive correlation of motility with in vitro invasion, whereas an inverse correlation was found for the degree of actin filament organization as well as for the number of vinculin plaques. Gap junctional communication was not directly related to in vitro invasiveness.

Stabilization of adhesion plaques by the expression of drebrin A in fibroblasts.

The expression of drebrin A was induced in mouse fibroblasts (L cells) after transformation of cells with a vector that carried cDNA for rat drebrin A (developmentally regulated brain protein A) under the control of the promoter of the gene for metallothionein-I. When drebrin was expressed in the transformed cells (MTI-5 cells), the organization of actin filaments changed such that stress fibers were converted to a mesh-like structure. After subsequent treatment with 5 micrograms/ml cytochalasin D (a reagent that depolymerizes actin filaments), MTI-5 cells maintained their shape, while cells of a drebrin-negative cell line, MTI-11, formed retraction processes. Simultaneously, actin filaments changed into patchy dot-like aggregates in the cytoplasm of both MTI-5 and MTI-11 cells. These aggregates are known as cytoplasmic pools. In MTI-5 cells, adhesion plaques that were resistant to treatment with cytochalasin D appeared upon expression of drebrin. These adhesion plaques were immunostained with vinculin-specific antibodies, while those in MTI-11 cells were hardly immunostained. The amount of vinculin in MTI-5 cells increased in parallel with increase in the level of drebrin. These results suggest that expression of drebrin A induces changes in the assembly of actin filaments and adhesion plaques, with resultant modulation of cellular adhesion to the substratum.

Flow cytometric analysis of isolated adult cardiomyocytes: vinculin and tubulin fluorescence during metabolic inhibition and ischemia.

Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.