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When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, alpha-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells' native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, alpha-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throughout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC6[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.

The localization of desmoplakins 1 and 2 (DP 1&2), components of desmosomes, vinculin, and actin, was studied in gingival epithelial cells grown on cell culture glass and on titanium plates with various surface topography. The results showed that epithelial cells attached and spread more readily on smooth than on rough, sandblasted titanium surfaces. Moreover, the cells appeared to develop more granular DP 1&2 immunoreactivity at their ventral surfaces when grown on smooth or etched titanium as compared to glass. In cells grown on sandblasted titanium surfaces, DP 1&2-specific immunoreactivity was primarily located at cell-cell contacts. Cells grown on smooth titanium surfaces harbored a fine network of actin filaments with apparent cell-to-cell organization. Vinculin was confined to cell-cell contact areas. No vinculin-containing focal adhesions could be detected, suggesting that the cells adhere either by means of close contacts, extracellular matrix contacts, or by means of hemidesmosomes. The findings suggest that smooth of finely grooved titanium surfaces could be optimal in maintaining the adhesion and specialized phenotype of gingival epithelial cells.

The extracellular localizations of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) were examined in cultured bovine capillary endothelial cells (BCEs) by an immunofluorescence method using BCEs treated with or without saponin and focal contact preparations. The specific immunofluorescence of cell surface uPA showed a patchy or strand-like distribution and was colocalized with vinculin strands indicating that uPA secreted from BCEs was mainly deposited at the cell surface of focal contacts. BCEs at a subconfluent density showed a higher intensity of specific immunofluorescence for uPA than when they were at a confluent density. tPA was observed over the dorsal surface of cultured BCEs and accentuated at their margins, suggesting that tPA was diffusely distributed on the luminal surface of BCEs in vivo. PAI-1 was distributed in the extracellular matrix under cultured BCEs. These findings suggest that uPA and PAI-1 are located under BCEs participating in the regulation of proteolytic activities provoked by plasminogen-PAs-plasmin system in vivo. The localization of tPA appears to be consistent with its function, which is to maintain the fluidity of the blood and to initiate thrombolysis in vivo.

The growth cone, the motile tip of developing neuronal processes, is considered responsible for the exact guidance of axons and synaptogenesis. High activity of tyrosine kinases in growth cones may contribute to the functions of growth cones. Our previous work revealed that vinculin is one of the endogenous substrates for intrinsic tyrosine kinases in the growth cone particle (GCP) fraction isolated from fetal rat brain. In the present study, we examined tyrosine phosphorylation and immunoblot analysis of vinculin in various fractions from fetal rat brains and adult synaptosomal fraction. Tyrosine phosphorylation of vinculin in the GCP fraction was more prominent than in any other fraction from fetal brain or synaptosomes from adult. Compared to other fractions, however, the enrichment of vinculin in the GCP fraction was not observed. Tyrosine phosphorylation of vinculin in the fraction was inhibited by genistein, a specific tyrosine kinase inhibitor. Although vinculin was also phosphorylated by protein kinase C in the GCP fraction, it incorporated a much smaller amount of 32P than MARCKS protein or GAP-43. The cytoskeletal subfraction from the GCP fraction contained a considerable amount of vinculin and it was one of the major substrates for tyrosine kinases in the GCP cytoskeleton. The membrane skeleton from the GCP fraction contained a low amount of vinculin but showed high kinase activity that phosphorylated vinculin. Taken together, our results suggest that tyrosine phosphorylation of vinculin contributes to the cytoskeletal organization of growth cones.

In this paper we have described the organization of F-actin and actin-binding proteins (ABP): alpha-actinin, myosin, tropomyosin, caldesmon, vinculin, talin, and spectrin, in differentiating astroglia in colony cultures. We observed that the microfilament (MF) network arrangements differ at various stages of astroglia development, but the composition of MF bundles and stress fibers is the same at all developmental stages. F-actin is closely colocalized with myosin, tropomyosin, caldesmon, and alpha-actinin. The striated pattern of myosin, tropomyosin, and caldesmon are superimposable. Tropomyosin and caldesmon extend along F-actin but are interrupted for short periods, whereas myosin is interrupted for longer periods. alpha-actinin colocalizes with tropomyosin and caldesmon but not with myosin. In astroglia at different stages of development spectrin is arranged in the form of fine networks spreading through the cell and does not follow the arrangement of MF bundles. Only F-actin, alpha-actinin, and vinculin can be detected at cell-cell junctions. In the areas of the focal contacts, F-actin, alpha-actinin, vinculin, and talin are present. They overlap each other, although talin and vinculin extend toward the cell membrane beyond F-actin and alpha-actinin. Astroglia undergo well-defined states of nonmotility, motility, and nonmotility again during differentiation. The changes in motility are paralleled by changes in the organization of F-actin and ABP: as GFAP-containing intermediate filaments increase in differentiating astroglia, the F-actin and ABP are down-regulated, leading to non motility.

Growth cones are intimately involved in determining the direction and extent of neurite elongation during development. They are able to monitor their environment and respond to it by undergoing directed motility. We have isolated a fraction enriched in growth cone particles from embryonic chick brain. Assayed by immunoblots, this fraction is enriched in GAP-43, and contains the cytoskeletal proteins actin, myosin II, neurofilament protein, tubulin, kinesin, and dynamin. All of the major components of focal adhesions are also present: alpha-actinin, vinculin, talin, and integrin. In addition to integrin, we also identify the cell adhesion molecules A-CAM, L1, fibronectin, and laminin in these particles. This preparation of isolated growth cone particles may be a useful model system for studying growth cone adhesion and motility.

Ectoplasmic specializations of Sertoli cells are actin containing structures found at sites of attachment to spermatids and to neighboring Sertoli cells. We suspect that these cytoskeletal structures are a form of actin-associated adhesion junction. If this is true, then molecular components, such as vinculin, that characterize actin-associated adhesion junctions in general should be present in ectoplasmic specializations. In this paper we have used two approaches to verify the prediction that vinculin is a component of ectoplasmic specializations. First, we have used fluorescence microscopy to probe immunologically for vinculin in ectoplasmic specializations associated with spermatids of the ground squirrel. Second, we have used immunogold techniques to probe for vinculin in ectoplasmic specializations of rat testis. Our results indicate that the immunological probe for vinculin was reactive with ectoplasmic specializations. In single label fluorescence experiments, linear patterns obtained with the vinculin probe were similar to those obtained with probes for filamentous actin. In double label experiments, the vinculin probe was co-distributed with the actin probes. In immunogold studies, specific labelling with the probe for vinculin occurred in ectoplasmic specializations both at sites of attachment to spermatids and adjacent to basal Sertoli cell junctions. Moreover, gold particles were concentrated adjacent to filament bundles within each ectoplasmic specialization. Our results support the conclusion that vinculin is present in ectoplasmic specializations. Further, they indicate that vinculin is co-distributed with actin bundles within each ectoplasmic specialization.

Interstitial cells of Leydig characteristically occur in clusters around blood vessels. Often these clusters remain intact when interstitial tissues are mechanically separated from other components of the testis. The presence of strong intercellular attachments is most likely one of the factors responsible for maintaining the integrity of Leydig cell clusters. In many tissues, actin associated adhesion junctions commonly provide intercellular attachment. To determine if actin associated adhesion junctions are present between Leydig cells, we have used 1) immunofluorescence to probe for two components that characterize these junctions in other tissues and 2) electron microscopy to examine areas of intercellular contact for evidence of microfilament related adhesion junctions. Isolated clusters of unsectioned cells, which had been fixed and detergent extracted, were probed with the F-actin specific strains rhodamine phalloidin and NBD-phallacidin and with an affinity purified primary antibody raised against human platelet vinculin. In regions of intercellular contact, fluorescence staining with the actin probes was intense and appeared as a solid linear band. Similar regions also stained with the vinculin probe. In double label experiments, actin and vinculin probes were co-distributed at sites of intercellular contact. Zones of intercellular contact, apparently similar to those detected with fluorescence microscopy, were observed at the ultrastructural level. At these sites, subsurface filaments, interpreted by us as actin, formed a dense carpet adjacent to the plasma membrane on each side of the junction. These filaments appeared to be organized into networks rather than discrete bundles.(ABSTRACT TRUNCATED AT 250 WORDS)

Resorption and migration phases alternate in the life of the osteoclast. We have previously described a specific microfilament structure at the attachment sites in resorbing osteoclasts. In the present study we have examined microfilaments and microtubules in both resorbing and migrating rat osteoclasts cultured on bone slices. In migrating osteoclasts microfilaments form so-called podosome structures containing vinculin, talin, and F-actin at the paramarginal area of the cell. When the osteoclast prepares itself for resorption, the podosomes gather to a certain area and form a broad ring around the area, which is then resorbed. In the resorbing osteoclast, vinculin and talin form a continuous double circle, which may be partially formed by podosomes, and between these double circles a broad zone is formed by F-actin. Narrow vinculin and F-actin rings were found in osteoclasts at the end of the resorption phase. The different configurations of microfilaments in 1 and 2 day cultures were correlated in terms of their relationship to the resorption lacunae. The vitamin A derivative isotretinoin significantly stimulated resorption and increased the number of microfilament configurations associated with the resorption pits. On the other hand, Bt2cAMP abolished resorption and prevented the formation of a specific ring structure of microfilaments. Based on these data, a kinetic model of the whole migration-resorption cycle of the osteoclast cultured on the bone slice is presented. With alpha-tubulin stainings of microtubules two different cytoskeletal organizations were observed. In migrating osteoclasts, microtubules were evenly distributed over the whole cell. In the resorbing osteoclast, there was a noticeable concentration of these cytoskeletal structures at cytoplasmic sites closest to the resorption lacuna. This orientation of microtubules may reflect the active secretory function of the resorbing osteoclast.

In this study, the distribution of free cholesterol in cholesterol-loaded endothelial cells was examined. For these studies, cell fractionation methods were used to assess marker enzyme activity and cholesterol distribution. Treatment of rabbit aortic endothelial cells for 3 days with 50 micrograms/ml of beta-very low density lipoprotein (beta-VLDL) or malondialdehyde-low density lipoprotein (MDA-LDL) but not LDL caused a 50-100% increase in total cell unesterified cholesterol. The accumulation of free rather than esterified cholesterol in endothelial cells may be due to the ratio of hydrolysis to esterification, which we have shown in this study to be 10-fold higher in endothelial cells than in smooth muscle cells. This free cholesterol is found in the fractions enriched in plasma membrane markers and, to a lesser extent, in the Golgi-enriched fractions. The amount of cholesterol per mg of protein was increased approximately 50% in these fractions from cells treated for 3 days with 50 micrograms/ml of beta-VLDL. These increases in cholesterol content were reversible upon incubation of cells for 3 days in medium containing 15% fetal bovine serum. Alterations in several membrane functions were also observed in cholesterol-loaded cells. The activity of alkaline phosphatase, an enzyme marker for plasma membranes, was decreased by 25% and an alteration in membrane-associated microfilaments was seen with phalloidin staining. This morphological change in microfilaments was reflected in a decrease in filament ends as shown by cytochalasin binding and occurred without a change in total actin or vinculin. These microfilament changes were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Polar axis formation and polar axis stabilization (or fixation) can be separated and analyzed in synchronously developing zygotes of the brown alga Fucus. Extensive experimental evidence points to a role for both the cytoskeleton and the extracellular matrix (ECM) in the process of axis fixation in Fucus. A structural complex composed of the cytoskeleton and the ECM has been postulated to stabilize membrane asymmetries generated as a result of axis-forming vectors. This axis stabilizing complex (ASC) may take the form of transmembrane connections between the cytoskeleton on the cytoplasmic face and the ECM on the external side of the plasma membrane, similar to focal contacts in animal cells. At present we know of two components in the proposed ASC of Fucus: an adhesive sulfated glycoprotein which is localized in the ECM, and an actin network which is localized on the adjoining cytoplasmic face. This preliminary report describes evidence for the presence of molecules in two-celled Fucus embryos that are similar to those found in focal contacts in animal cells, i.e. vinculin, integrin and vitronectin. However, their localization and interaction with each other relative to the polar axis has yet to be determined. These initial observations will provide the basis to pursue further an analysis of these components in the process of polar axis fixation.