Influence of polylysine on adhesion of fibroblasts to glass substrates visualized by total internal reflection microscopy

The cell adhesion topography of mouse fibroblasts growing on glass substrates has been investigated. In order to compare cell adhesion on covered and uncovered glass, substrates were partly exposed to a solution with 0.1 mg/ml polylysine (300 kDa) for 15 min before incubation with cell suspension. After cultivation for 1, 3, 6, and 24 h their adhesion was visualised by total internal reflection microscopy. In the presence of polylysine, cells incubated for 1 h were strongly attracted to the substrate, leading to a typical cell adhesion topography characterised by round concavities under the ventral cell membrane with an approximate diameter of 1 μm. The cavity-surrounding rims were tightly bound to the glass surface. During further cell cultivation, the topography changed into a well-organised adhesion pattern with focal contact areas on the periphery of the cells. In contrast to the polylysine-mediated adhesion, cells growing on untreated surfaces did not exhibit the cavity-like topography at any stage of cultivation, but a more point spread adhesion with a dense clustering of contact-forming areas.     

Biomimetic implant coatings

Biomaterials and tissue engineering technologies are becoming increasingly important in biomedical practice, particularly as the population ages. Cellular responses depend on topographical properties of the biomaterial at the nanometer scale. Structures on biomaterial surfaces are used as powerful tools to influence or even control interactions between implants and the biological system [; ]. The influence of nanometer sized surface structures on osteoblastlike cell interactions was tested with niobium oxide coatings on polished titanium slices (cp-Ti grade 2). The aim of the study was to investigate the influence of nanoscopic surface structures on osteoblast interactions in order to support collagen I production and cell adhesion. The coatings were done by means of the sol-gel process. The surface structure was adjusted by annealing of the metaloxide ceramic coatings due to temperature depended crystal growth. The applied annealing temperatures were 450, 550 and 700 degrees C for 1 h, corresponding to Ra-numbers of 7, 15 and 40 nm. The surfaces were characterized by means of AFM, DTA/TG, diffractometry and white light interferometry. The cell reactions were investigated concerning adhesion kinetics, migration, spreading, cell adhesion, and collagen I synthesis. The smooth surface (Ra=7 nm) resulted in the fastest cell anchorage and cell migration. The closest cell adhesion was reached with the surface structure of Ra=15 nm. The roughest surface (Ra=40 nm) impedes the cell migration as well as a proper spreading of the cells. The best results concerning cell adhesion and spreading was reached with an intermediate surface roughness of Ra=15 nm of the niobium oxide coating on cp-titanium slices.     

Saturated C21–C33Hydrocarbons Are Involved in the Self-Regulation of Pseudomonas fluorescensAdhesion to a Glass Surface

One of the two putative groups of antiadhesins was identified in Pseudomonas fluorescensby the method of gas chromatography–mass spectrometry. A mixture of high-molecular unbranched hydrocarbons (HC) with a chain length from 21 to 33 carbon atoms reduced cell adhesion to a glass surface. These HC accumulated in the culture liquid to a total concentration of 10–15 g/l; the concentrations of individual HC ranged from 0.1 to 3.0 g/l. After the addition of individual HC to the bacterial culture, the number of cells attached to the glass surface decreased. This decrease in cell adhesion was due to the enhanced aggregation of the bacterial cells, which promoted mechanical (hydrodynamic) cell detachment from the surface.     

MANGANESE STIMULATES ADHESION AND SPREADING OF MOUSE SARCOMA I ASCITES CELLS

Adhesion of Sarcoma I cells (SaI) to untreated or to serum-treated glass was examined by layering ⁵¹Cr-labeled cells on the substrate for 20 min at 34°C and determining the glass-bound radioactivity after the monolayers were rinsed. Adhesion to untreated glass proceeded in sodium chloride-imidazole-potassium medium (SIK) without added divalent cations, whereas SaI adhered maximally to the serum-coated substrate only in the presence of 50 µM or more Mn. Divalent Mg, Ca, Co, Ni, or Zn were inactive or minimally active. Mn-stimulated adhesion was sharply temperature dependent, reversible upon removal of Mn, and inhibited by Ca as well as by cytochalasin B, vinblastine, or tetracaine. Adhesion of SaI in SIK did not ensue when cells or the coated substrate were pretreated with Mn and washed in SIK before the adhesion assays. Microscope observations showed that Mn induced the formation of cell processes, ruffles, and veils, and that SaI spread on the uncoated or serum-coated substrate when exposed to Mn. Cells withdrew veils and processes and rounded up when postincubated in Mn-free medium. Formation of cell processes and spreading was inhibited by cytochalasin B, vinblastine, or tetracaine. Manganese-induced adhesion seems to require the participation of microtubules and microfilaments and may be mediated by an effect of Mn on Ca fluxes. The results support the role of cell processes and spreading in cell-to-substrate adhesion.     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

Isolation and characterization of an adherent, 8-azaguanine resistant variant of the Burkitt lymphoma cell line, Raji

A substrate adherent, fibroblast-like cell line (Raji-A) has been isolated from a suspension culture of an established Burkitt lymphoma cell line (Raji). Except for the altered morphology associated with substrate attachment, Raji-A is identical to Raji with respect to karyotype, isozyme composition, susceptibility to Epstein-Barr virus (EBV) infection and inducibility of latent EBV. In order to facilitate fusion experiments with Raji-A, drug resistant variants were induced by treating cells with ethylmethane sulfonate followed by selection in growth medium which contains 8-azaguanine. Three clones (AGRO, AGR3 and AGR6) were found to be resistant to 50 μg/ml of 8-azaguanine. They had only 10–15% as much hypoxanthineguanine phosphoribosyltransferase activity as wild type cells and a low plating efficiency of 1–3 × 10⁻⁶ in the selective medium, HAT. Potential uses of these variants for studying EBV-lymphoblastoid cell interactions as well as human genetics by cell hybridization are discussed.     

Urokinase receptor (CD87) clustering in detergent-insoluble adhesion patches leads to cell adhesion independently of integrins

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidyl inositol-anchored protein that mediates cell adhesion to the extracellular matrix protein vitronectin (VN). We demonstrate here that this cell adhesion process is accompanied by the formation of an adhesion patch characterized by an accumulation of uPAR into areas of direct contact between the cell and the matrix. The adhesion patch requires the glycolipid anchor and develops only on a VN-coated substrate, but not on fibronectin. It consists of detergent-insoluble microdomains that accumulate F-actin and tyrosine-phosphorylated proteins, but not β₁ integrins. Lack of inhibition of adhesion in the presence of integrin-blocking reagents and adhesion on a VN fragment without the RGD sequence indicated that the adhesion of uPAR-bearing cells on VN could occur independently of integrins. Hence, uPAR-mediated cell adhesion on VN relies on the formation of a unique cellular structure that we have termed “detergent-insoluble adhesion patch” (DIAP).     

Changes induced by concanavalin A in morphological and adhesive properties of suspension cells

In the presence of concanavalin A obtained from nata beans (Canavalia gladiata), mastocytoma cells, Ogun, HR-1, Janosky and monocytic leukemia cells in suspension culture rapidly adhered to the glass surface and gradually spread their cytoplasms like monolayer cells. The morphological shape of the spreading cells differed according to cell strains. The spreading cells were not detached by the treatment with trypsin or with EDTA. Actinomycin D and cycloheximide did not affect either the adhesion or the spreading of mastocytoma cells. Colchicine inhibited the adhesion of mastocytoma cells only slightly but it caused a great change in the morphological shape of the spreading cells. The cell adhesion was temperature-dependent and was inhibited markedly by -mannose and α-methyl- -glucoside and slightly by -galactose.     

The removal of extracellular fibronectin from areas of cell-substrate contact

Immunofluorescent labeling for fibronectin was largely excluded from sites of closest contact between spreading chicken gizzard fibroblasts and the substratum. This was observed by double immunofluorescent labeling of fixed cells for fibronectin and vinculin, a smooth muscle intracellular protein that is specifically associated with focal adhesion plaques, in conjunction with interference-reflection microscopy. When the cells were plated on a fibronectin-coated substratum they adhered to its surface and rapidly spread on it. The immunofluorescent labeling for fibronectin in those cultures (after fixation and triton permeabilization) was usually absent from the newly formed, vinculin-containing focal adhesion plaques. We have found, however, that the accessibility to the cell-substrate gap at the focal adhesion plaques is limited and therefore a more direct approach was adopted. We have found that cells spreading on a substrate coated with rhodamine-labeled fibronectin progressively removed the underlying protein from the substrate. The removal of fibronectin involved at least two distinct mechanisms. Part of the substrate-associated fibronectin was removed from small areas and displaced toward the cell center. The arrowhead-shaped areas from which fibronectin was removed often coincided with vinculin-rich focal contacts. We observed, however, many areas where focal contacts were found over unperturbed fibronectin carpet, as well as fibronectin-free areas with no overlapping focal contacts. The possibilities that fibronectin is actively displaced from areas of cell-substrate contact, that the focal adhesion plaques are transiently associated with these areas and their implications on the dynamics of cell spreading and locomotion are discussed. The second route of fibronectin removal from the substrate was endocytosis. The rhodamine-labeled fibronectin was found in the cells in a partial or transient association with clathrin-containing structures.     

Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line

R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [¹²⁵I]-PACAP or [¹²⁵I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC₅₀ of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC₅₀ in agreement with the pharmacological profile of the VPAC₁ receptor subtype: PACAP = VIP > [K¹⁵, R^16, L²⁷]VIP(1–7)/GRF(8–27) = [R¹⁶]ChSn (two VPAC₁ agonists) HELODERMIN = secretin. RO 25–1553, a selective activator of VPAC₂ receptor was inactive at 1 μM. Dose-response curves of VPAC₁ agonist molecules (PACAP, VIP, [K¹⁵, R¹⁶, L²⁷]VIP(1–7)/GRF(8–27), [R¹⁶]ChSn) were shifted to the right by the VPAC₁ receptor antagonist [AcHis¹, D-Phe², Lys¹⁵, Leu¹⁷]VIP(3–7)/GRF(8–27), with a K_i of 3 ± 1 nM (n = 3). The presence of VPAC₁ receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC₁ receptors underwent homologous and heterologous desensitization.

This study provides the first evidence for the expression of functional VPAC₁ receptors undergoing rapid desensitization in HEL cells.     

CGRP antagonists and capsaicin on celiac ganglia partly prevent postoperative gastric ileus

The role of capsaicin-sensitive pathways and CGRP in postoperative gastric ileus was investigated. Abdominal surgery was performed under enflurane anesthesia, and 5 min later, the 20-min rate of gastric emptying was measured by the phenol red method in conscious rats. Surgery inhibited gastric emptying by 76–83% compared with rats receiving anesthesia alone. Capsaicin on the celiac/mesenteric ganglia (10–21 days before) reduced gastric ileus by 33 ± 8%, whereas perivagal capsaicin had no effect. The IV CGRP-induced inhibition of gastric emptying was completely reversed by the CGRP antagonist, CGRP(8–37) (30 μg, IV); CGRP(8–37) (15, 30, or 60 μg) or CGRP monoclonal antibody #4901 (2 mg protein) decreased the inhibition of gastric emptying by 11 ± 7%, 51 ± 13%, 47 ± 3%, and 45 ± 17%, respectively. These results indicate that CGRP and splanchnic capsaicin-sensitive afferents are involved in mediating part of the gastric ileus observed immediately after abdominal surgery.     

NOV/CCN3 Induces Adhesion of Muscle Skeletal Cells and Cooperates with FGF2 and IGF-1 to Promote Proliferation and Survival

During mammalian development, expression of the Nephroblastoma overexpressed gene (NOV/CCN3) is tightly regulated in skeletal muscles. Ex vivo, ectopic expression of NOV blocks myogenic differentiation. NOV also supports endothelial cell adhesion and angiogenesis through interactions with integrins. Integrins play fundamental roles during myogenesis. In this study, we show that NOV mediates adhesion and spreading of myoblasts. Myoblasts adhesion to NOV does not require proteoglycans and is dependent on integrin β1, whereas spreading involves another RGD-sensitive integrin. The C-Terminal part of NOV as well as full-length is able to support adhesion of myoblasts; in addition, both increase focal-adhesion kinase (FAK) phosphorylation. Furthermore, NOV is an adhesive substrate that, combined with FGF2 or IGF-1, promotes cell specific proliferation and survival, respectively, in a better way than fibronectin. Taken together, these results identify NOV as an adhesion substrate for myoblasts which, in concert with growth factors, could play a role in the physiology of muscle cells.     

Absence of Filamin A Prevents Cells from Responding to Stiffness Gradients on Gels Coated with Collagen but not Fibronectin

Cell types from many tissues respond to changes in substrate stiffness by actively remodeling their cytoskeletons to alter spread area or adhesion strength, and in some cases changing their own stiffness to match that of their substrate. These cell responses to substrate stiffness are linked to substrate-induced changes in the state, localization, and amount of numerous proteins, but detailed evidence for the requirement of specific proteins in these distinct forms of mechanical response are scarce. Here we use microfluidics techniques to produce gels with a gradient of stiffness to show the essential function of filamin A in cell responses to mechanical stimuli and dissociate cell spreading and stiffening by contrasting responses of a pair of human melanoma-derived cell lines that differ in expression of this actin cross-linking protein. M2 melanoma cells null for filamin A do not alter their adherent area in response to increased substrate stiffness when they link to the substrate only through collagen receptors, but change adherent area normally when bound through fibronectin receptors. In contrast, filamin A-replete A7 cells change adherent area on both substrates and respond more strongly to collagen I-coated gels than to fibronectin-coated gels. Strikingly, A7 cells alter their stiffness, as measured by atomic force microscopy, to match the elastic modulus of the substrate immediately adjacent to them on the gradient. M2 cells, in contrast, maintain a constant stiffness on all substrates that is as low as that of A7 cells on the softest gels examined (1000 Pa). Comparison of cell spreading and cell stiffening on the same gradient substrates shows that cell spreading is uncoupled from stiffening. At saturating collagen and fibronectin concentrations, adhesion of M2 cells is reduced compared to that of A7 cells to an extent approximately equal to the difference in adherent area. Filamin A appears to be essential for cell stiffening on collagen, but not for cell spreading on fibronectin. These results have implications for different models of cell protrusion and adhesion and identify a key role for filamin A in altering cellular stiffness that cannot be compensated for by other actin cross-linkers in vivo.     

An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3- mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation

The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well-defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface-bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required.     

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.     

Determination of transdermal sildenafil in nude mouse skin by reversed-phase high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of sildenafil transdermal permeation of nude mouse skin. A reversed-phase column with UV detection at 224 nm was used for chromatographic separation. The mobile phase consisted of 32% acetonitrile with 0.2% phosphoric acid in water at pH 5.3 adjusted with 10 M NaOH with the flow-rate set at 1.0 ml/min. The limit of quantitation achieved was 5 ng/ml, and the calibration curve showed good linearity over the concentration range of 5–500 ng/ml. The relative standard deviations of within- and between-day analyses were all within 15%. Sildenafil was found to be stable between pH 3 and 12 during 24-h incubation with skin. After transdermal administration of 15.8 μg/ml of sildenafil to nude mouse skin, it was detected as early as 15 min. The transport amount of sildenafil could be quantitated and, at pH 8–11, had the highest permeation rate in nude mouse skin.     

Spread of fast thermowaves through the cerebral cortex produced by visual stimulation

Thermowaves spreading through the cerebral cortex were observed and investigated during acute experiments on white rats using a new technique — that of thermoencephaloscopy (TES) through the intact skull. These waves were induced by regular visual stimulation (at intervals of 1.5–3 min) or by initiating one of the flashes and spread through the cortex along trajectories of five basic types; amplitude of thermowaves: 0.005–0.1°C; length: 10–15 mm; duration 1.2–11.4 sec; velocity: 1–33 mm/sec; extent of pathway: 2–56 mm. They appeared with a high degree of probability (of 0.92) during the interval between 15 sec before and 26 sec after the flash. Numbers of moving waves declined by the point of stimulus application and rose by 7–8 sec after the flash. Waves arose in 50% of cases in the contralateral visual cortex (areas 17 and 18a), spreading to the midline and crossing to the ipsilateral hemisphere (areas 17, 18a, and 7). Local waves moving along a circular trajectory were also discovered in the contralateral visual cortex. Several types of wave, differing in trajectory, also arose in the ipsilateral visual cortex. Mechanisms and possible significance of this effect are examined.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 467–475, July–August, 1989.     

Microspike-mediated particle transport towards the cell body during early spreading of 3T3 cells

Freshly trypsinized 3T3 cells send out microspikes of 0.2 μm diameter and up to 10 μm length within 20 min after attachment to a glass substratum. The microspikes move actively and eventually attach to the substratum. Subsequently, lamellae flow out between lines of attached microspikes. If, however, colloidal gold particles of 0.2–0.4 μm diameter and clusters of gold particles up to 4 μm in diameter are placed on the substratum and a microspike attaches to them, we observed two reactions of the microspikes to this contact. They either retract upon contact, transporting the attached particles to the cell surface at a speed of 0.2 μm/sec, or the particles flow toward the cell body while the microspike stays in place. This action results in the clearing of a circular area around each spreading cell before lamellae flow out. “Clearing” proceeds at serum concentrations between 1 and 20% and in concentrations of colchicine up to 20 μm/ml. In concentrations of cytochalasin B higher than 5 μg/ml, however, particle removal is completely inhibited, although the microspikes are still produced by the cell. Transmission electron microscopy shows that the microspikes contain mostly longitudinally oriented microfilaments and only a few microtubules, if any.     

Changes in fibroblast contractility, morphology, and adhesion in response to a phorbol ester tumor promoter

We have studied the effects of the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the contractility, locomotion, morphology, and adhesion of two mammalian fibroblastic cell lines. Using the silicone rubber substratum technique, we have found that the first observable response to the tumor promoter is a rapid weakening of cell contractility (8-15 min). This is followed by gradual morphological changes, characterized by a hyperextension of the cells' leading lamellae, which stretch out to an unlimited degree, and occasionally even detach from the cell bodies. Treated cells also become able to crawl onto hydrophobic substrata which are insufficiently adhesive to support the spreading of untreated fibroblasts. We suggest that both the hyperextension and the ability to spread on nonadhesive surfaces can be explained as consequences of the reduced contractility, and that this reduced contractility may also help to explain the increased invasiveness and loss of anchorage dependence by transformed cells.     

Focal adhesion disassembly is an essential early event in hepatic stellate cell chemotaxis

Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF.