phalloidine and tetramethylrhodamine-isothiocyanate

Corneal stem cell niches are located within the limbus of the eye and are believed to play an important role in corneal regeneration. These niches are often lost in corneal disease or trauma. Our work explores the design of artificial limbal stem cell niches by the fabrication of biodegradable electrospun rings containing bespoke microfeatures. In creating artificial niches, we seek to provide a physically protective environment for limbal cells to act as a cell reservoir for tissue regeneration purposes. This study describes the first step in this challenge to produce structures which structurally approximate to the limbal niches. This was achieved using a combination of electrospinning and microfabrication. Initial microfabricated structures were developed using microstereolithography via a layer-by-layer photocuring approach based on the patterning of photocurable polymers, in this case polyethylene glycol diacrylate. This was then used as a template on which to electrospin a biodegradable membrane of poly(lactic-co-glycolic acid) 50:50, which incorporates the features of the underlying microfabricated structures. The study describes preliminary evaluation of these constructs using rabbit limbal epithelial and stromal cells.

Topics: Animals; Cell Adhesion; Cell Proliferation; Epithelial Cells; Limbus Corneae; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microtechnology; Phalloidine; Rabbits; Rhodamines; Tissue Engineering; Tissue Scaffolds; Wound Healing

In the current study we examined if the multifunctional cytokine TGF-beta1 mediated glucose-evoked increases in connexin-43(Cx43)-mediated intercellular communication in cells of the human collecting duct (HCD).. RT-PCR and western blot analysis were used to confirm mRNA and protein expression of TGF-beta1 and Cx43 in HCD-cells. The effect of TGF-beta1 and high glucose (25 mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-beta1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca2+-microfluorimetry.. At 24 hrs, high glucose (25 mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-beta1 (2 ng/ml) at low glucose (5 mM). Both high glucose and TGF-beta1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-beta. Furthermore, high glucose-evoked changes were inhibited by a TGF-beta1-specific monoclonal antibody. Mannitol (25 mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-beta1 evoked changes in Cx43 expression were biphasic. An early (4-8 hr) transient decrease in expression was followed by an increase in protein expression (12-24 hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24 hrs coincided with a TGF-beta1 specific increase in touch-evoked transmission of Ca2+-signals between coupled cells.. High glucose evoked a TGF-beta1 mediated increase in Cx43 expression and gap-junction mediated cell-cell communication in HCD-cells. These changes may maintain epithelial integrity of the collecting duct following hyperglycaemic assault as observed in diabetes.

Topics: Actins; Cell Communication; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Connexin 43; Cytosol; Dose-Response Relationship, Drug; Fluorescent Dyes; Glucose; Humans; Immunohistochemistry; Kidney Tubules, Collecting; Phalloidine; Proteins; Rhodamines; RNA, Messenger; Simian virus 40; Transforming Growth Factor beta1; Up-Regulation

Mycophenolic acid (MPA) is a potent inhibitor of the inosine monophosphate dehydrogenase and used as an immunosuppressive drug in transplantation. MPA inhibits proliferation of T- and B-lymphocytes by guanosine depletion. Since fibroblasts rely on the de novo synthesis of guanosine nucleotides, it is assumed that MPA interacts with fibroblasts causing an increased frequency of wound healing problems. We show a downregulation of the cytoskeletal proteins vinculin, actin and tubulin in fibroblasts exposed to pharmacological doses of MPA using microarray technology, real-time polymerase chain reaction (PCR) and Western blot. This reduction in RNA and protein content is accompanied by a substantial rearrangement of the cytoskeleton in MPA-treated fibroblasts as documented by immunofluorescence. The dysfunctional fibroblast growth was validated by scratch test documenting impaired migrational capacity. In contrast, cell adhesion was increased in MPA-treated fibroblasts. The results of the cultured human fibroblasts were applied to skin biopsies of renal transplant recipients. Skin biopsies of patients treated with MPA expressed less vinculin, actin and tubulin as compared to control biopsies that could explain potential wound healing problems posttransplantation. The perspective of MPA-induced cytoskeletal dysfunction may go beyond wound healing disturbances and may have beneficial effects on (renal) allografts with respect to scarring.

Topics: Biopsy; Carbocyanines; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Dermatologic Surgical Procedures; Dose-Response Relationship, Drug; Fibroblasts; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Immunohistochemistry; Immunosuppressive Agents; Indoles; Mycophenolic Acid; Phalloidine; Rhodamines; Skin

The disturbance of plasma membrane carbohydrates and of lipopolysaccharide (LPS) ligands in relation to cytoskeletal transformations of haemocytes has been investigated after chronic exposure of pond snails (Lymnaea stagnalis) to the peroxidizing toxicant fomesafen. Neither of the two lectins used (concanavalin A and wheat germ agglutinin) showed any binding modification after incubation of the snails in the presence of the toxicant. However, after exposure of the snails to fomesafen, a clear and persistent reduction in LPS labelling of haemocytes occurred. The actin cytoskeleton of the same cells also appeared to be sensitive to the toxicant. The reduction in LPS-binding sites was related to actin staining, leading to the hypothesis that LPS ligands and actin could be similarly modulated by the toxicant. Damaged cells showed non-adherent membrane portions with reduced filopodial extrusions, exhibiting a smooth surface free of microvilli. These changes could lower the spreading and adhesion of the cells and could therefore account for the loss in their phagocytic capabilities.

Topics: Actins; Animals; Benzamides; Cell Survival; Dose-Response Relationship, Drug; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Hemocytes; Herbicides; Lectins; Lymnaea; Phagocytosis; Phalloidine; Rhodamines; Time Factors; Water Pollutants, Chemical

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC50 of 88.6+/-5.3 microM and a t0.5 of 89.7+/-7.2 min, with 50 microM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 microM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 microM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4+/-3.6% and 41.0+/-15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.

Topics: Actins; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Survival; Clotrimazole; Cytoskeleton; Dose-Response Relationship, Drug; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fructose-Bisphosphate Aldolase; Glucose; Growth Inhibitors; Humans; Lactic Acid; Phalloidine; Phosphofructokinase-1; Rhodamines; Streptavidin

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Blood Platelets; Cell Adhesion; CHO Cells; Cricetinae; Cricetulus; Enzyme Activation; Fibrinogen; Fibrinolysin; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Focal Adhesion Protein-Tyrosine Kinases; Humans; Kinetics; Ligands; Microscopy, Fluorescence; Mutation; Phalloidine; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Conformation; rhoA GTP-Binding Protein; Rhodamines; Transfection

Talin plays a key role in the assembly and stabilisation of focal adhesions, but whether it is directly involved in force transmission during morphogenesis remains to be elucidated. We show that the traction force of Dictyostelium cells mutant for one of its two talin genes talB is considerably smaller than that of wild-type cells, both in isolation and within tissues undergoing morphogenetic movement. The motility of mutant cells in tightly packed tissues in vivo or under strong resistance conditions in vitro was lower than that of wild-type cells, but their motility under low external force conditions was not impaired, indicating inefficient transmission of force in mutant cells. Antibody staining revealed that the talB gene product (talin B) exists as small units subjacent to the cell membrane at adhesion sites without forming large focal adhesion-like assemblies. The total amount of talin B on the cell membrane was larger in prestalk cells, which exert larger force than prespore cells during morphogenesis. We conclude that talin B is involved in force transmission between the cytoskeleton and cell exterior.

Topics: Actins; Animals; Cell Membrane; Cell Movement; Chimera; Dictyostelium; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Focal Adhesions; Microscopy, Confocal; Morphogenesis; Mutation; Myosins; Phalloidine; Protein Structure, Tertiary; Protozoan Proteins; Rhodamines; Talin

In order to advance our knowledge of the nitrergic nervous system in flatworms, the patterns of the NADPH-diaphorase (NADPH-d) reaction and cGMP immunoreactivity, after stimulation with a nitric oxide donor in the presence of an inhibitor of phosphodiesterase, were investigated in cercaria of Diplostomum chromatophorum. This is the first time the presence of NADPH-d activity has been detected in a larval fluke, and the first time the presence of cGMP immunoreactivity has been detected in a flatworm. The NADPH-d reaction occurs in the ventral sucker, the hind body and the tail. cGMP immunoreactivity was detected in the muscle cells of the body and in two pairs of sensory cells at the anterior end of the body and in the middle of the furca. The sensory cells also showed 5-HT immunoreactivity. The spatial relationship between the cGMP and the 5-HT immunoreactivities was studied. By staining with TRITC-labelled phalloidin, the pattern of the muscle fibres was revealed.

Topics: Actins; Animals; Cyclic GMP; Histocytochemistry; Immunohistochemistry; Life Cycle Stages; NADPH Dehydrogenase; Nitric Oxide; Nitric Oxide Donors; Phalloidine; Rhodamines; Serotonin; Staining and Labeling; Trematoda

Dd-TRAP1 is a Dictyostelium homologue of TRAP-1, a human protein that binds to the type 1 tumor necrosis factor (TNF) receptor. TRAP-1 has a putative mitochondrial localization sequence and shows significant homology to members of the HSP90 family. Although TRAP-1 is mainly localized to mitochondria in several mammalian cells, in certain tissues it is also localized at specific extramitochondrial sites. In Dictyostelium cells, Dd-TRAP1 is predominantly located in the cell membrane/cortex during growth and just after starvation. Double staining of vegetatively growing cells with the anti-Dd-TRAP1 antibody and TRITC-phalloidin has demonstrated colocalization of Dd-TRAP1 and F-actin at the leading edge of cortical protrusions such as pseudopodes. Coupled with differentiation, however, Dd-TRAP1 located at the cortical region is translocated to mitochondria in spite of the absence of the mitochondrial localization sequence at its N-terminus. The translocation of this protein raises interesting and fundamental questions regarding possible mechanisms by which Dd-TRAP1 is involved in cellular differentiation.

Topics: Actins; Amino Acid Sequence; Animals; Cell Differentiation; Cell Membrane; Conserved Sequence; Culture Media, Conditioned; Dictyostelium; HSP90 Heat-Shock Proteins; Hydrogen-Ion Concentration; Mitochondria; Molecular Sequence Data; Phalloidine; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins; Rhodamines; Sequence Alignment; Subcellular Fractions; Transformation, Genetic

To investigate the role of dynamic changes in actin cytoskeleton in cellular response to oxidative stress, we have analyzed the state of actin polymerization and synthesis in human alveolar cells exposed to paraquat, an oxidant agent. Cellular content of monomeric actin was assayed by DNase I inhibition. It decreased significantly in treated cells and depended on paraquat concentration. Paraquat treatment of cells caused an increase of the filamentous pool of actin and a parallel decrease of the monomeric one. Such shift was shown to be irreversible. SDS-PAGE of cytoskeletal fractions was performed under reducing and non-reducing conditions. No cross-linking of actin monomers to form large aggregates appeared to be related to the observed paraquat-induced increase of the filamentous actin pool. Morphological analyses by indirect immunofluorescence and ultrastructural examination confirmed the presence of microfilaments in treated cells. Conventional bundles of filaments were not observed, but numerous single filaments appeared dispersed within the cytoplasm. Pulse-chase experiments showed a strong increase of de novo synthesis of actin in treated cells, whereas actin degradation rate remained unaffected. In conclusion, the different approaches lead to a concordant picture of cellular response to oxidant stress at the level of the actin filament system. Actin pools are modified: the overall number of filaments increases, whereas the monomeric species decreases. As a result of the shift of actin from the monomeric pool to the filamentous one, the de novo synthesis of actin is increased.

Topics: Actins; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Herbicides; Humans; Paraquat; Phalloidine; Pulmonary Alveoli; Rhodamines; Tumor Cells, Cultured