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At least 19 recordsLinked to original sources

PubMed2001

Eicosanoids participate in the regulation of cardiac glucose transport by contribution to a rearrangement of actin cytoskeletal elements.

Intact actin microfilaments are required for insulin-regulated glucose transporter isoform 4 (GLUT4) translocation to the plasma membrane. Lipoxygenase (LO) metabolites have recently been shown to contribute to the regulation of actin cytoskeleton rearrangement. In the present investigation, ventricular cardiomyocytes were used to study the effects of two structurally different LO inhibitors (esculetin and nordihydroguaiaretic acid) on insulin signalling events, glucose uptake, GLUT4 translocation and the actin network organization. Insulin stimulation increased glucose uptake 3-fold in control cells, whereas LO inhibition completely blocked this effect. This was paralleled by a slight reduction in the insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2. However, inhibition of 12-LO did not affect the association of phosphatidylinositol 3-kinase with IRS-1 and the phosphorylation of Akt/protein kinase B in response to insulin. Addition of 12(S)-hydroxyeicosatetraenoic acid almost completely restored the insulin action in cells exposed to nordihydroguaiaretic acid. Insulin stimulation increased cell surface GLUT4 2-fold in control cells, whereas LO inhibition abrogated the insulin-stimulated GLUT4 translocation. LO inhibition induced a prominent disassembly of actin fibres compared with control cells. In conclusion, we show here that 12(S)-hydroxyeicosatetraenoic acid plays a role in the organization of the actin network in cardiomyocytes. LO inhibition blocks GLUT4 translocation without affecting downstream insulin signalling. These data suggest that LO metabolites participate in the regulation of glucose transport by contributing to a rearrangement of actin cytoskeletal elements.

Actins↗

PubMed1997

Uptake of tetraphenylporphycene and its photoeffects on actin and cytokeratin elements of HeLa cells.

In the present work we have continued our studies in the photobiological properties of the 2,7,12,17-tetraphenylporphycene (TPPo). In particular, the uptake, the subcellular localization and the photoeffects on two cytoskeletal elements (actin, microfilaments and cytokeratin intermediate filaments) of HeLa cells have been analyzed. The uptake kinetics of TPPo, determined by fluorescence spectroscopy, was initially very rapid, reaching saturation at approximately 6 h of incubation. This porphycene tends to be accumulated mainly in rounded particles distributed throughout the cytoplasm. The morphological comparison of the localization pattern of TPPo and those of acridine orange and rhodamine 123, which are fluorescence markers for lysosomes and mitochondria respectively, allowed us to confirm that this porphycene is mainly accumulated in lysosomal organelles. The results obtained after treatment with TPPo and red light indicated that this compound is very effective in mediating the photodestruction of lysosomes. The photosensitizing effects on the cytoskeletal elements studied depended on both the irradiation time and the elapsed time after treatment. The implications of damage to lysosomes and actin and cytokeratin filaments on the process of cell death is discussed.

Acridine Orange↗

PubMed1999

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Actins↗

PubMed2000

Smooth muscle alpha-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum response factor-containing activation complex.

Previous studies have shown that multiple serum response factor (SRF)-binding CArG elements were required for smooth muscle cell (SMC)-specific regulation of smooth muscle (SM) alpha-actin expression. However, a critical question remains as to the mechanisms whereby a ubiquitously expressed transcription factor such as SRF might contribute to SMC-specific expression. The goal of the present study was to investigate the hypothesis that SMC-selective expression of SM alpha-actin is due at least in part to (1) unique CArG flanking sequences that distinguish the SM alpha-actin CArGs from other ubiquitously expressed CArG-dependent genes such as c-fos, (2) cooperative interactions between CArG elements, and (3) SRF-dependent binding of SMC-selective proteins to the CArG-containing regions of the promoter. Results demonstrated that specific sequences flanking CArG B were important for promoter activity in SMCs but not in bovine aortic endothelial cells. We also provided evidence indicating that the structural orientation between CArGs A and B was an important determinant of promoter function. Electrophoretic mobility shift assays and methylation interference footprinting demonstrated that a unique SRF-containing complex formed that was selective for SMCs and, furthermore, that this complex was probably stabilized by protein-protein interactions and not by specific interactions with CArG flanking sequences. Taken together, the results of these studies provide evidence that SM alpha-actin expression in SMCs is complex and may involve the formation of a unique multiprotein initiation complex that is coordinated by SRF complexes bound to multiple CArG elements.

Actins↗

PubMed2002

Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based peroxisome motility in plants.

Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect.

Actins↗

PubMed1997

POLLEN GERMINATION AND TUBE GROWTH.

Many aspects of Angiosperm pollen germination and tube growth are discussed including mechanisms of dehydration and rehydration, in vitro germination, pollen coat compounds, the dynamic involvement of cytoskeletal elements (actin, microtubules), calcium ion fluxes, extracellular matrix elements (stylar arabinogalactan proteins), and control mechanisms of gene expression in dehydrating and germinating pollen. We focus on the recent developments in pollen biology that help us understand how the male gamete survives and accomplishes its successful delivery to the ovule of the sperm to effect sexual reproduction.

Journal Article↗

PubMed2004

Actin and microtubules in cell motility: which one is in control?

The cytoskeleton is composed of three distinct elements: actin microfilaments, microtubules and intermediate filaments. The actin cytoskeleton is thought to provide protrusive and contractile forces, and microtubules to form a polarized network allowing organelle and protein movement throughout the cell. Intermediate filaments are generally considered the most rigid component, responsible for the maintenance of the overall cell shape. Cytoskeletal elements must be coordinately regulated for the cell to fulfill complex cellular functions, as diverse as cell migration, cell adhesion and cell division. Coordination between cytoskeletal elements is achieved by signaling pathways, involving common regulators such as the Rho guanosine-5'-triphosphatases (GTPases). Furthermore, evidence is now accumulating that cytoskeletal elements participate in regulating each other. As a consequence, although their functions seem well defined, they are in fact overlapping, with actin playing a role in membrane trafficking and microtubules being involved in the control of protrusive and contractile forces. This cytoskeletal crosstalk is both direct and mediated by signaling molecules. Cell motility is a well-studied example where the interplay between actin and microtubules appears bidirectional. This leads us to wonder which, if any, cytoskeletal element leads the way.

Actins↗

PubMed2002

Tensegrity behaviour of cortical and cytosolic cytoskeletal components in twisted living adherent cells.

The present study is an attempt to relate the multicomponent response of the cytoskeleton (CSK), evaluated in twisted living adherent cells, to the heterogeneity of the cytoskeletal structure--evaluated both experimentally by means of 3D reconstructions, and theoretically considering the predictions given by two tensegrity models composed of (four and six) compressive elements and (respectively 12 and 24) tensile elements. Using magnetic twisting cytometry in which beads are attached to integrin receptors linked to the actin CSK of living adherent epithelial cells, we specifically measured the elastic CSK response at quasi equilibrium state and partitioned this response in terms of cortical and cytosolic contributions with a two-component model (i.e., a series of two Voigt bodies). These two CSK components were found to be prestressed and exhibited a stress-hardening response which both characterize tensegrity behaviour with however significant differences: compared to the cytosolic component, the cortical cytoskeleton appears to be a faster responding component, being a less prestressed and easily deformable structure. The discrepancies in elastic behaviour between the cortical and cytosolic CSK components may be understood on the basis of prestress tensegrity model predictions, given that the length and number of constitutive actin elements are taken into account.

Actins↗

PubMed1995

One of three CCArGG box/serum response elements of the beta-actin gene is an insulin-responsive element.

The cytoskeletal actins are abundant proteins in mammalian nonmuscle cells. We have previously reported that physiological concentrations of insulin induced beta-actin transcription in rat H4 hepatoma cells. To define whether one or more of the three CCArGG box elements or other elements within the beta-actin gene promoter is an insulin response element, we transfected H4 cells with regions of the human beta-actin gene promoter fused to the chloramphenicol acetyltransferase gene. A 350-basepair DNA fragment was isolated that mediates both insulin and serum effects. This fragment contains at least two up-stream elements, a CCAAT box and a CCArGG box, and accounts for more than 70% of the basal activity of the beta-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum induction, suggesting a difference in their mechanisms of action. Mutation of the CCAAT box drastically reduced basal expression, with no effect on insulin induction. In contrast, a mutation of the CCArGG element reduced basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulated the activity, but not the binding, of a factor(s) that associates with the CCArGG box. These data demonstrate that in H4 cells, insulin induction of beta-actin gene expression was mediated at least in part through one of the three beta-actin CCArGG elements.

Actins↗

PubMed1989

Actin cytoskeletal lesions in differentiated human colon carcinoma Caco-2 cells after exposure to soybean agglutinin.

We have investigated the effects of soybean agglutinin on the cytoskeletal element actin in differentiated Caco-2 cells. The actin cytoskeleton of the cells was visualized by fluorescence microscopy using 7-nitrobenz-2-oxa-1, 3-diazole phallacidin as a specific marker for F-actin. Compared with control Caco-2 cells no changes in the fluorescence pattern were observed after incubation with soybean agglutinin. However, using the deoxyribonuclease-I inhibition assay a dose-related response was noted in the increase of intracellular G-actin after a 2-hour incubation period with soybean agglutinin. Already after exposure for 15 min to soybean agglutinin a decrease in intracellular F-actin was demonstrable. This apparent depolymerization could be prevented by incubating the Caco-2 cells with soybean agglutinin and the appropriate monosaccharide simultaneously. The increase in the amount of G-actin appeared to be correlated with a shortening of microvilli on the Caco-2 cells.

Actins↗

PubMed1988

Distribution of microtubules and other cytoskeletal filaments during myotube elongation as revealed by fluorescence microscopy.

Distribution of microtubules and other cytoskeletal filaments in growing skeletal muscle cells (myotubes) was studied in vitro by fluorescence microscopy using fluorescin-labeled antibodies and phalloidin, a specific antiactin drug. In the distal elongating tips of myotubes, microtubules were the major cytoskeletal elements; actin and intermediate filaments were much less abundant. On the other hand, colcemid- and nocodozole-treatments caused disruption of microtubles and also prompt retraction of growth tips to form myosacs, a type of deformed myotube. Actin filaments remained unaffected during the retraction. The difference in the distribution of the 3 cytoskeletal filaments in the region of growth tips was most remarkable in the case of those myotubes in the process of recovery from myosacs. In an early phase of recovery, the cellular processes extending from myosacs were enriched with both microtubules and intermediate filaments, but not with actin filaments. Later, when the processes became further developed, intermediate filaments were scarce at the extreme ends. Fluorescein-labeled actin introduced by a micro-injection method was minimally incorporated into filaments in the cellular processes. We conclude that microtubules make up the cytoskeletal element which is most responsible for elongation or spreading of growth tips of myotubes in vitro.

Actins↗

PubMed1999

Mammalian chondrocytes expanded in the presence of fibroblast growth factor 2 maintain the ability to differentiate and regenerate three-dimensional cartilaginous tissue.

The differentiated phenotype of chondrocytes from hyaline cartilage is gradually lost during expansion in monolayers. Chondrocytes can reexpress their differentiated phenotype by transfer into an environment that prevents cell flattening, but serially passaged cells never completely recover their chondrogenic potential. We report that chondrocytes expanded (up to 2000-fold) in the presence of fibroblast growth factor 2 (FGF-2) dedifferentiated, but fully maintained their potential for redifferentiation in response to environmental changes. After seeding onto three-dimensional polymer scaffolds, chondrocytes expanded in the presence of FGF-2 formed cartilaginous tissue that was histologically and biochemically comparable to that obtained using primary chondrocytes, in contrast to chondrocytes expanded to the same degree but in the absence of FGF-2. The presence of FGF-2 inhibited the formation of thick F-actin structures, which otherwise formed during monolayer expansion, were maintained during tissue cultivation, and were associated with reduced ability of chondrocytes to reexpress their differentiated phenotype. This study provides evidence that FGF-2 maintains the chondrogenic potential during chondrocyte expansion in monolayers, possibly due to changes in the architecture of F-actin elements and allows more efficient utilization of harvested tissue for cartilage tissue engineering.

Actins↗

PubMed2005

Poly(A)+ RNA and cytoskeleton during cyst formation in the cap ray of Acetabularia peniculus.

The configuration and distribution of polyadenylated RNA (poly(A)+ RNA) during cyst formation in the cap rays of Acetabularia peniculus were demonstrated by fluorescence in situ hybridization using oligo(dT) as a probe, and the spatial and functional relationships between poly(A)+ RNA and microtubules or actin filaments were examined by immunofluorescence microscopy and cytoskeletal inhibitor treatment. Poly(A)+ RNA striations were present in the cytoplasm of early cap rays and associated with longitudinal actin bundles. Cytochalasin D destroyed the actin filaments and caused a dispersal of the striations. Poly(A)+ RNA striations occurred in the cytoplasm of the cap rays up to the stage when secondary nuclei migrated into the cap rays, but they disappeared after the secondary nuclei were settled in their positions. At that time, a mass of poly(A)+ RNA was present around each of the secondary nuclei and accumulated rRNA. This mass colocalized with microtubules radiating from the surface of each secondary nucleus and disappeared when the microtubules were depolymerized by butamifos, which did not affect the configuration of actin filaments. These masses of poly(A)+ RNA continued to exist even after the cap ray cytoplasm divided into cyst domains. Thus two distinct forms of poly(A)+ RNA population, striations and masses, appear in turn at consecutive stages of cyst formation and are associated with distinct cytoskeletal elements, actin filaments and microtubules, respectively.

Acetabularia↗

PubMed2002

Wound healing recapitulates morphogenesis in Drosophila embryos.

The capacity to repair a wound is a fundamental survival mechanism that is activated at any site of damage throughout embryonic and adult life. To study the cell biology and genetics of this process, we have developed a wounding model in Drosophila melanogaster embryos that allows live imaging of rearrangements and changes in cell shape, and of the cytoskeletal machinery that draws closed an in vivo wound. Using embryos expressing green fluorescent protein (GFP) fusion proteins, we show that two cytoskeletal-dependent elements -- an actin cable and dynamic filopodial/lamellipodial protrusions -- are expressed by epithelial cells at the wound edge and are pivotal for repair. Modulating the activities of the small GTPases Rho and Cdc42 demonstrates that these actin-dependent elements have differing cellular functions, but that either alone can drive wound closure. The actin cable operates as a 'purse-string' to draw the hole closed, whereas filopodia are essential for the final 'knitting' together of epithelial cells at the end of repair. Our data suggest a more complex model for epithelial repair than previously envisaged and highlight remarkable similarities with the well-characterized morphogenetic movement of dorsal closure in Drosophila.

Actins↗

PubMed1989

Comparative growth dynamics and actin concentration between cultured human myofibroblasts from granulating wounds and dermal fibroblasts from normal skin.

The normal contraction of open wounds and many forms of pathologic contracture are related by the presence of a contractile fibroblast known as a myofibroblast. The function of this cell has been postulated as a result of previous pharmacological, immunological, and biochemical testing on strips of contracted connective tissue. The purpose of this study was to develop a specific assay that could measure the concentration of one contractile element (actin) within cultured myofibroblasts isolated from a contracting wound and in normal fibroblasts from uninjured dermis. Rates of growth and actin concentration through 15 days of culture were compared among populations of paired control fibroblasts from normal dermis and granulating wound myofibroblasts from three patients. Growth curves showed that myofibroblasts always grew slower than fibroblasts. An enzyme-linked immunosorbent assay showed that actin concentration was generally greater in mass cultures of granulating wound myofibroblasts than in fibroblasts from uninjured dermis. During exponential growth (1-6 days) the average actin concentration among myofibroblast lines ranged from 24 to 62 pg/cell. Average actin levels among control fibroblasts ranged from 3 to 47 pg/cell during the same interval. After 15 days of culture, actin concentration peaked twice. The first actin peak occurred within the period of exponential growth. At confluency, cellular actin levels dropped. Superconfluent cultures exhibited a second actin peak that displayed an irregular pattern of actin concentration. The latter observation suggested an artifact that might be the result of three-dimensional matrix of cells that altered points of cell adhesion and produced an irregular pattern of actin concentration. These data show that the phenotype of increased actin in cultured myofibroblasts was carried over by myofibroblasts from contracted skin wounds to culture. Because of a higher concentration of actin in myofibroblasts than in undifferentiated fibroblasts, these data suggest that the differentiation process of myofibroblasts may be associated with an increased availability of monomeric actin for filament synthesis. This study demonstrates that the use of tissue culture and our enzyme-linked immunosorbent assay will be a useful method to study factors affecting myofibroblast phenotypic modulation. Future studies should be directed toward developing procedures for isolation of pure populations of myofibroblasts as well as extracellular matrices that would maintain the morphology of both differentiated myofibroblasts and normal undifferentiated fibroblasts.

Actins↗

PubMed1997

Roles of microfilaments and intermediate filaments in adrenal steroidogenesis.

The problem for the steroidogenic cell if it is to accelerate steroid synthesis in response to trophic stimulation, consists in moving cholesterol from the sites of synthesis and storage to mitochondria at an accelerated rate. The most intensely studied situation is that in which the sterol is stored as ester in lipid droplets. Cholesterol ester must be de-esterified and transported to mitochondria where steroid synthesis begins. Since droplets and mitochondria are now known to be attached to intermediate filaments and since these structures are not contractile, it appears to be necessary to invoke the actions of other cytoskeletal elements. Actin microfilaments are involved in cholesterol transport so that it is tempting to propose that the contractile properties of actomyosin are used in this process. It is known that an energy-dependent contractile process involving actin is capable of disrupting intermediate filaments. Since the intermediate filaments appear to act by keeping lipid droplets and mitochondria apart, disruption of the filaments accompanied by a contractile process would be expected to allow these two structures to come together. This would open the way for the transfer of cholesterol to the steroidogenic pathway. This should be regarded as a first step. The events necessary for entry of cholesterol from droplets into the mitochondria remain to be clarified. In addition, the transport process for newly synthesized cholesterol that is not stored in droplets, is still not understood. At least four protein kinase enzymes have been identified in the cytoskeletons of adrenal cells, namely, Ca2+/calmodulin-dependent kinase, protein kinase (Ca2+ and phospholipid-dependent), myosin light chain kinase, and protein kinase A (cyclic AMP-dependent). The Ca2+/calmodulin kinase promotes transport of cholesterol to mitochondria and does so under conditions in which phosphorylation of vimentin and myosin light chain occurs. Phosphorylation of vimentin results in disruption of intermediate filaments while phosphorylation of light chain promotes contraction of the actomyosin ring. It now appears that intermediate filaments are cross-linked by actin filaments so that such contraction would be expected to produce significant structural changes in the cytoskeleton and the attached organelles. Although the details of the changes taking place in the organ in vivo are not known, the potential for interaction between droplets and mitochondria as the result of these changes in intermediate filaments and actomyosin, is clear. Protein kinase C is activated by ACTH and cyclic AMP, although this activation does not appear to be directly involved in the regulation of steroid synthesis. Nevertheless, vimentin is a substrate for this enzyme, and changes in the organisation of vimentin filaments and the attached organelles under the influence of protein kinase C have been reported in other cells. Presumably these changes represent part of the response to ACTH because when protein kinase C is activated by phorbol ester, the cytoskeletal changes necessary for rounding up take place but such changes are not accompanied by increased steroid synthesis. Protein kinase A causes rounding of adrenal cells. and cytoskeletons. This kinase also causes increased cholesterol transport and, hence, stimulation of steroid synthesis. The enzyme also causes phosphorylation of vimentin but with a different cytoskeletal reorganisation from that seen with the other three kinase enzymes. Clearly phosphorylation plays a major role in these responses. Phosphorylation alters the morphology and the functions of the cytoskeleton and this, in turn, is associated with accelerated cholesterol transport. It is now necessary to define the details of the specific phosphorylation reactions that occur during the response to ACTH, that is, which amino acids are phosphorylated and to what extent by each of the kinase enzymes.

Actin Cytoskeleton↗

PubMed2005

Bile acid deoxycholate induces differential subcellular localisation of the PKC isoenzymes beta 1, epsilon and delta in colonic epithelial cells in a sodium butyrate insensitive manner.

Elevated levels of bile acids have been implicated in the abnormal morphogenesis of the colonic epithelium thus contributing to colorectal cancer (CRC). Alternatively sodium butyrate (NaB) produced by anaerobic fermentation of dietary fibre is regarded as being protective against colon cancer. Bile acids such as deoxycholic acid (DCA) are thought to mediate some of their actions by differentially activating protein kinase C (PKC). We examined the effects of DCA on the subcellular localisation of PKC-beta(1), -epsilon and -delta and whether these responses could be modulated by NaB. HCT116 cells endogenously express PKC-epsilon and -delta but not PKC-beta. DCA treatment results in endogenous PKC-epsilon translocation but not PKC-delta after 1 hr. To study the subcellular localisation of PKC isoforms in response to DCA in real time, PKC-beta(1), PKC-epsilon and PKC-delta functionally intact green fluorescent protein (GFP) fusion constructs were used. Stimulation with 300 microM DCA induces rapid translocation of PKC-beta(1)-GFP and PKC-epsilon-GFP but not PKC-delta-GFP from the cytosol to the plasma membrane in 15 min. Interestingly, pretreatment with 4mM NaB does not modify the response of the PKC isoenzymes to DCA as PKC-beta(1)-GFP and PKC-epsilon-GFP translocates to the plasma membrane in 15 min whereas PKC-delta-GFP localisation remains unaltered. Immunofluorescence shows that PKC-beta(1)-GFP and PKC-epsilon-GFP cells treated with DCA colocalise with the cytoskeletal elements actin and tubulin adjacent to the plasma membrane. Our findings demonstrate that the differential activation of the PKC isoenzymes by DCA may be of critical importance for the functional responses of colonic epithelial cells. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

Butyrates↗

PubMed1982

Synthesis and distribution of cytoskeletal elements in endothelial cells as a function of cell growth and organization.

Confluent cultures of adult bovine aortic endothelial (ABAE), corneal endothelial (BCE), and fetal bovine heart endothelial (FBHE) cells form a monolayer of highly flattened, closely apposed, and nonoverlapping cells. In ABAE and BCE cultures, this is associated with a 50-fold decrease in the rate of DNA sythesis and correlates with a 14-fold decrease in protein synthesis. In contrast, in confluent FBHE cultures only partial decreases in the rates of DNA synthesis (6-fold) and protein synthesis (3-fold) are observed. FBHE cells therefore fulfill the morphological, but not the biochemical, criteria for confluent cultured endothelial cell monolayers. The appearance of the cytoskeletal elements actin, tubulin, and vimentin in sparse and confluent cultures of endothelial cells has been analyzed by two-dimensional gel electrophoresis and immunofluorescence. Sparse versus confluent ABAE, FBHE, and BCE cultures showed no changes in their relative rates of synthesis or cellular content of tubulin. Actin behaved similarly to tubulin in FBHE and BCE cultures, while in ABAE cultures a small increase (3-fold) in its relative rate of synthesis was observed in confluent versus sparse cultures. BCE cultures showed no change in the rate of synthesis of vimentin, but the cellular content of vimentin was markedly increased when cultures reached confluence. When the distribution of vimentin in both sparse and confluent BCE cultures was analyzed by immunofluorescence, in both cases it appeared distributed throughout the cytoplasm as thin fibers and bundles of fibers. In confluent ABAE cultures, both the relative amount and biosynthetic rate of vimentin increased by 15-fold. This increase in the intracellular accumulation of vimentin correlated with its immunofluorescent distribution within the cells. While in sparse cultures, vimentin appeared to be distributed as thin fibers, in confluent cultures thick curl-like fibrous bundles could be seen distributed throughout the cytoplasm and organized in a perinuclear ring. In contrast, in FBHE cultures no significant changes in the distribution and organization or rate of synthesis of vimentin were observed.

Actins↗