Distribution of filipin-sterol complexes on cultured muscle cells: cell- substratum contact areas associated with acetylcholine receptor clusters

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylcholine receptor clusters identified by fluorescence labeled alpha-bungarotoxin. Freeze-fracture of the cells confirmed these observations. The membrane in these areas was highly convoluted and contained aggregates of large P-face intramembrane particles (probably representing acetylcholine receptors). If cells were fixed and then treated with the sterol- specific antibiotic filipin before fracturing, the pattern of filipin- sterol complex distribution closely followed the pattern of cell- substratum contact. Filipin-sterol complexes were in low density in the regions where the membrane contained clustered intramembrane particles. These membrane regions were away from the substratum (bright white areas in reflection interference contrast; depressions of the P-face in freeze-fracture). Filipin-sterol complexes were also in reduced density where the membrane was very close to the substratum (dark areas in reflection interference contrast; bulges of the P-face in freeze- fracture). These areas were not associated with clustered acetylcholine receptors (aggregated particles). This result suggests that filipin treatment causes little or no artefact in either acetylcholine receptor distribution or membrane topography of fixed cells and that the distribution of filipin-sterol complexes may closely parallel the microheterogeneity of membranes that exist in living cells.     

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.     

Locomotion of sponges and its physical mechanism

Active locomotion by individual marine and freshwater sponges across glass, plastic and rubber substrata has been studied in relation to the behavior of the sponges' component cells. Sequential tracing of sponge outlines on aquarium walls shows that sponges can crawl up to 160 microns/hr (4 mm/day). Time-lapse cinemicrography and scanning electron microscopy reveal that moving sponges possess distinctive leading edges composed of motile cells. Sponge locomotion was found to be mechanically similar to the spreading of cell sheets in tissue culture both with respect to exertion of traction (which causes the wrinkling of rubber substrata) and with respect to the patterns of adhesive contacts formed with the substratum (as observed by interference reflection microscopy). Other similarities include the orientation of sponge locomotion along grooves and the preferential extension onto more adhesive substrata. Neither the patterns of wrinkling produced in rubber substrata nor the distributions of adhesive contacts seen by interference reflection microscopy show evidence of periodic, propagating waves of surface contractions, such as would be expected if the sponges' mechanism of locomotion were by peristalsis or locomotory waves. Our observations suggest that the displacement of sponges is achieved by the cumulative crawling locomotion of the cells that compose the sponge's lower surface. This mode of organismal locomotion suggests new explanations for the plasticity of sponge morphology, seems not to have been reported from other metazoans, and has significant ecological implications.     

Adhesion of cells to protein carpets: do cells' feet have to be black?

In most physiological situations, cell contact with a substratum is mediated by proteins of extracellular matrix. Therefore, an increasing number of cell-substratum adhesion studies employ substrata covered with one or more proteins of extracellular matrix. To visualize the most adhesive cell structures, focal contacts and focal adhesions, the interference reflection microscopy has been widely used. It has been generally accepted that these strongly adhesive structures can be seen as black streaks in interference reflection microscopy. Calculations are presented herein, which although simplified, suggest that when cells are plated on protein-covered substrata, their focal contacts may not always appear black in interference reflection microscopy.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

Costameres are sites of force transmission to the substratum in adult rat cardiomyocytes

Costameres, the vinculin-rich, sub-membranous transverse ribs found in many skeletal and cardiac muscle cells (Pardo, J. V., J. D. Siciliano, and S. W. Craig. 1983. Proc. Natl. Acad. Sci. USA. 80:363-367.) are thought to anchor the Z-lines of the myofibrils to the sarcolemma. In addition, it has been postulated that costameres provide mechanical linkage between the cells' internal contractile machinery and the extracellular matrix, but direct evidence for this supposition has been lacking. By combining the flexible silicone rubber substratum technique (Harris, A. K., P. Wild, and D. Stopak. 1980. Science (Wash. DC). 208:177-179.) with the microinjection of fluorescently labeled vinculin and alpha-actinin, we have been able to correlate the distribution of costameres in adult rat cardiac myocytes with the pattern of forces these cells exert on the flexible substratum. In addition, we used interference reflection microscopy to identify areas of the cells which are in close contact to the underlying substratum. Our results indicate that, in older cell cultures, costameres can transmit forces to the extracellular environment. We base this conclusion on the following observations: (a) adult rat heart cells, cultured on the silicone rubber substratum for 8 or more days, produce pleat-like wrinkles during contraction, which diminish or disappear during relaxation; (b) the pleat-like wrinkles form between adjacent alpha-actinin-positive Z-lines; (c) the presence of pleat-like wrinkles is always associated with a periodic, "costameric" distribution of vinculin in the areas where the pleats form; and (d) a banded or periodic pattern of dark gray or close contacts (as determined by interference reflection microscopy) has been observed in many cells which have been in culture for eight or more days, and these close contacts contain vinculin. A surprising finding is that vinculin can be found in a costameric pattern in cells which are contracting, but not producing pleat-like wrinkles in the substratum. This suggests that additional proteins or posttranslational modifications of known costamere proteins are necessary to form a continuous linkage between the myofibrils and the extracellular matrix. These results confirm the hypothesis that costameres mechanically link the myofibrils to the extracellular matrix. We put forth the hypothesis that costameres are composite structures, made up of many protein components; some of these components function primarily to anchor myofibrils to the sarcolemma, while others form transmembrane linkages to the extracellular matrix.     

Competition of contacting heterotypic epithelial sheets for the territory in culture

Co-cultured epithelial cells of various lines formed mixed cohesive sheets. Contact interactions of different cells in the sheets were studied by phase contrast and interference reflection microscopy, time-lapse microcinematography, transmission and scanning electron microscopy. It was found that heterotypic cells in the combined sheets were locked together by the complexes of specialized contacts and were metabolically coupled, as shown by the dye transfer. At the same time heterotypic cells continued to extend pseudopods at the contacting lateral surfaces and to attach these pseudopodies to substratum. Due to competition of pseudopod attachments one group of cells in the sheet often progressively pushed another group from the substratum. This competition of cells for substratum may provide an important mechanism for morphogenetic reorganisation of epithelial sheets and of other groups of interacting cells in vivo.     

Surface proteins of the gliding bacterium Cytophaga sp. strain U67 and its mutants defective in adhesion and motility.

Surface proteins of the gliding bacterium Cytophaga sp. strain U67 that make contact with glass substrata were radioiodinated, using a substratum-immobilized catalyst (Iodo-Gen). At least 15 polypeptides were iodinated, fewer than the number labeled by surface biotinylation of whole cells; these polypeptides define the set of possible candidates for the surface protein(s) that mediates gliding-associated substratum adhesion. The labeling of three adhesion-defective mutants exhibited two characteristic patterns of surface iodination which involved addition, loss, or alteration of several polypeptides of high molecular weight. An adhesion-competent revertant of mutant Adh3 and one of Adh2 exhibited the wild-type labeling pattern. Two other Adh2 revertants resembled their adhesion-defective parent. The labeling pattern of surface polypeptides of a nongliding but adhesive cell strain was similar to that of the wild type.     

Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells

Surface protrusions at the leading edge of a moving cell that make contact with the surrounding extracellular matrix (ECM) are its main motor for locomotion and invasion. Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) form specialized membrane rosette-shaped contact sites on planar substrata as shown by interference reflection microscopy (IRM). Such activity is lacking in normal cells. These rosette contacts are more labile than other adhesion sites, such as focal and close contacts. Ultrastructural studies demonstrate that rosettes are sites at which membrane protrusions from the ventral cell surface contact the substratum. These protrusions are filled with meshworks of microfilaments and contain the pp60src oncogene product, actin, vinculin, and alpha-actinin. However, unlike focal contacts, at the rosettes these proteins interact to extend a highly motile membrane. Rosettes have the biological activity of degrading ECM components, as demonstrated by (1) local degradation of fibronectin substrata at sites of rosette contacts, but not focal and close contacts; (2) localization of putative antiprotease antibody at sites of rosette contacts, but not at focal an close contacts; and (3) local disruption of fibronectin matrix at sites of protrusive activity seen by transmission electron microscopy (TEM). In addition, formation of the rosette contact is insensitive to the ionophore monensin, and to inhibitors of proteolytic enzymes, while local fibronectin degradation at rosette contacts is inhibited by inhibitors of metalloproteases, 1,10-phenanthroline and NP-20. I consider these membrane protrusions of the rosette contacts in RSV-transformed cells specialized structural entities--invadopodia--that are involved in the local degradation of the ECM.     

Actomyosin organization in stationary and migrating sheets of cultured human endothelial cells

We used immunofluorescence microscopy to study the organization of actin, myosin and vinculin in confluent endothelial cells and in cells migrating into an experimental wound and interference reflection microscopy to assess the cell-substratum adhesion pattern in these cells. In confluent stationary endothelial cell monolayers actin showed a distinct cell-to-cell organization. Myosin, on the other hand, was diffusely distributed and was clearly absent from cell peripheries. Vinculin was confined as linear arrays to cell-cell contact areas. Interference reflection microscopy revealed areas of close and distant adhesion but no focal adhesion sites in these cultures. Twelve hours after experimental wounding a distinct zone of advancing cells was seen at the wound edge. These cells showed a spreadout morphology and, in contrast to stationary cells, had a stress fibre-type organization of both actin and myosin. Vinculin was in the migrating cells seen as plaques at the ventral cell surface. In interference reflection microscopy numerous focal adhesions were seen. The results indicate that the actomyosin system forms the structural basis for monolayer organization of endothelial cells and responds by reorganization upon cell migration.     

Microtubules, microfilaments and adhesion patterns in differentiating chick retinal pigment epithelial (RPE) cells in vitro

The distribution of microtubules (MT), microfilaments (MF), and patterns of cell-to-substratum adhesion were studied by tubulin antibody labeling, NBD-phallacidin staining and by reflection interference contrast (RIC) microscopy respectively in colonies of differentiating RPE cells obtained from explants after 10 days in culture. In each colony three zones could be identified: a central zone of packed well-differentiated cuboidal cells (zone 1), an intermediate zone of more flattened, pleomorphic cells (zone 2) and a peripheral zone of very spread cells at the edge of the colony (zone 3). As visualized with antibodies to tubulin, the MT distribution in cells of each zone was distinctly different and correlated well with differences in cell shape. Changes in the distribution of MF were more striking. In the cuboidal well-differentiated cells of zone 1, prominent cortical bands but no stress fibers were observed after staining with NBD-phallacidin and RIC microscopy showed that the cells lacked strong adhesion to the substratum. Stress fibers, in addition to cortical bands of MF, were seen in the more spread, less differentiated cells of zone 2 and focal contacts were observed when these cells were examined by RIC microscopy. The flattened least differentiated cells in zone 3 lacked cortical bands but had prominent stress fibers. These cells displayed a variety of adhesion forms ranging from a mosaic of far and close contacts to numerous focal contacts and broad focal adhesions. Our results show that as the RPE cells display less differentiated morphologies, i.e. are more flattened and less densely packed towards the edge of the colony, there is a gradual decrease in the cortical bands of MF and an increase in the number and prominence of stress fibers. This increase in numbers of stress fibers is correlated with an increase in the cell adhesiveness to the substratum, as estimated by RIC microscopy. These results strongly support the general observation that normal epithelial cells in colonies tend to adhere to the substratum more strongly by marginal cells than by the more differentiated centrally located cuboidal cells which have well developed intercellular contacts.     

Loss of focal contacts accompanies the density dependent inhibition of cell growth

The formation of focal contacts by fibroblast-like 3T3 and 9390 cells and endothelial XTH-2 cells was examined with the use of reflection interference contrast microscopy at different cell densities. It was found that cells density-inhibited in growth loose all focal contacts with the substratum. This observation explains the disappearance of stress fibers in density-inhibited cells, and also offers an explanation for the mechanism of density-dependent inhibition of proliferation of anchorage-dependent cells.     

A new type of substratum adhesion structure in NRK cells revealed by correlated interference reflection and electron microscopy

The substratum adherent membrane of NRK cells in vitro has been studied using correlated interference reflection and surface replica electron microscopy. Structures visualized by interference reflection microscopy were identified in the subsequently prepared platinum-carbon replicas of the adherent membranes. Substratum adherent membranes were prepared using a hypotonic shock/cell shearing technique (lysis-squirting). Typically 20% of the original ventral membrane surface area and 50% of the original focal adhesion number were retained. Microfilament bundle termini, clathrin-coated sheets and pits, cytotic vesicles having a ridged surface and groups of membrane associated particles were well preserved in the replicas. Two types of isolated adhesion structures were found after lysis-squirting. In addition to typical elongate focal adhesions containing actin and vinculin, we report the existence of adherent membrane patches lacking microfilament bundles and negative for vinculin labelling, but coated with clathrin and identifiable in interference reflection microscopy as less dark than focal adhesions and having dot, U- or sinusoidal shapes.     

The distribution of fibronectin in attachment sites of chick fibroblasts

Primary chick heart fibroblasts were cultured on glass coverslips and examined by reflection contrast microscopy and then by indirect immunofluorescence microscopy using affinity purified antifibronectin antibody. Fibronectin was found to be primarily localized in areas of ‘close’ contact and in intensely staining fibrous webs that were usually external to the cytoplasmic matrix. Focal contacts, in which there is intimate contact between the cell and the substratum by localized attachment, failed to react with the anti-fibronectin antibody despite a variety of extraction procedures designed to increase the accessibility of antibody to this type of attachment site. It is concluded that in chick fibroblasts, fibronectin participates in cell attachment at areas of close contact and fibrillar attachment sites, but is often excluded from the attachment pads of focal contacts.     

Cell-to-substrate adhesions during spreading and locomotion of carcinoma cells : A study by microcinematography and reflection contrast microscopy

Motility and patterns of adhesion were determined by time-lapse cinematography and reflection contrast microscopy for two types of carcinoma cells, selected for their different motile behavior and not for their malignancy. Cells from the V2 rabbit carcinoma become locomotory soon after having established the necessary contact to the substratum. In contrast, cells from a human epidermoid carcinoma (LICR-OC-1) first attain a fully spread configuration before some cells slightly round up again for a slow locomotory activity of short range and duration. Reflection contrast showed that during spreading and locomotion, the cells from both carcinomas displayed a predominance of grey, the color associated with close contacts. Fully spread cells, on the other hand, presented a multitude of focal contacts in individually different arrangements of black streaks and dots, randomly distributed over the entire cell area. The functional meaning of this heterogeneity in the arrangement of focal contacts in fully spread cells is not yet understood. The importance of close contacts for spreading and locomotion, however, seems to be established and is in agreement with findings reported for other cell types engaged in the same activities. It is therefore suggested that the formation of substrate contacts depends on cellular activity rather than on the cell type.     

Substratum attachment of embryonic mesoderm cells in culture

The cell-substratum adhesive characteristics of cultured chick embryo primary mesoderm cells have been examined by interference reflection microscopy and transmission electron microscopy under various conditions. Correlations were drawn between the type of adhesion and the degree of motility shown by the cells. During the rapid spreading and motility of cells cultured on fibronectin-containing substrata, focal contacts (10 to 15-nm gap) were rare and close contacts (about 30-nm gap) were predominant. By contrast, when the cells were immobile, after 5 d in culture, extensive focal contacts were present, together with stress fibers. The results indicate that tight cell-substratum contact is incompatible with rapid cell motility and that fibronectin acts by inducing the formation of close contacts rather than focal contacts.     

Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures

The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference- reflection microscopy and thus represented circular forms of close cell- substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.     

Substrate Contact,Mucus, and Eugregregarine Gliding1

ABSTRACT. Experiments have been carried out on Gregarina garnhami to examine some of the factors that may be significant in gliding. Suspension of gregannes in Ficoll showed that substrate contact is essential. Reflection interference microscopy shows that there are fluctuating surface/substrate contacts, but it is not necessary for the whole of the undersurface of the cell to be in close contact with the substrate. Gliding is always accompanied by the formation of a mucus trail. The effects of the drugs amphetamine and ephedrine on mucus trail formation and gliding have been examined. Lateral undulations of the epicyte folds have previously been implicated in grcgarinc gliding, but G. garnhami does not exhibit lateral undulations of the epicyte folds as it moves. The folds are predominantly straight with indications of varicosities or swellings along the length of the folds. These observations are discussed in relation to gliding movement.     

Fibroblast adhesion to RGDS shows novel features compared with fibronectin

As previously shown by others, the fibroblast attachment and spreading activity of fibronectin is mimicked by a short peptide (RGDS or longer) from the cell binding domain. Normal rat kidney fibroblasts showed similar attachment kinetics on either peptide GRGDSC or bovine plasma fibronectin and binding to either substratum was inhibited by peptide alone. We now demonstrate, however, considerable differences in biological activity between peptide and fibronectin. In particular, cells developed novel adhesion structures on peptide-coated substrata. Interference reflection microscopy showed a predominance of small round dark grey/black patches of adherent membrane ("spots") with relatively few focal adhesions, which occurred only at the outermost cell margins in contrast to their distribution in cells spread on fibronectin. The spots were resistant to detergent extraction and stained less strongly or not at all for vinculin. Electron microscopy in vertical thin section showed that the ventral surface of the cell was characterized by "point-contacts", corresponding in size to the spot structures seen by interference reflection microscopy, and which were only occasionally associated with microfilaments. Cells also required a higher substratum loading of peptide than fibronectin to promote spreading and proceeded to spread less rapidly and to a lesser extent, developing very few and extremely fine actin cables.