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PubMed1998

Purification and characterization of autophagosomes from rat hepatocytes.

To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-l-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediate-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1, cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).

Animals↗

PubMed2005

Arfs, phosphoinositides and membrane traffic.

Arf (ADP-ribosylation factor) GTP-binding proteins function in cells to regulate membrane traffic and structure. Arfs accomplish this task through modification of membrane lipids and the recruitment of proteins, including coat proteins and actin, to membrane surfaces. Arf1 and Arf6 are the most divergent and most studied human Arf proteins that localize predominantly to the Golgi complex and plasma membrane respectively. We have been studying the targeting of Arf1 and Arf6 to these specific compartments and the common and divergent activities that they exert on these membranes. We have found that Arf6 acts through activation of type I phosphatidylinositol 4-phosphate 5-kinases to generate phosphatidylinositol 4,5-bisphosphate and that this activity is instrumental in facilitating the actin cytoskeletal rearrangements and alterations in endosomal membrane trafficking observed with increased Arf6 activation. Arf1 can also stimulate the activity of phosphatidylinositol kinases and recruit coat proteins and actin cytoskeletal elements to the Golgi complex.

ADP-Ribosylation Factor 1↗

PubMed2002

Eicosanoids and the regulation of cardiac glucose transport.

Intact actin microfilaments are necessary for insulin-regulated GLUT4 translocation from intracellular pools to the plasma membrane. Products of the lipoxygenase (LO) pathway were shown to be implicated in the regulation of actin cytoskeleton rearrangement. The aim of this study was to examine the role of these LO products for cardiac insulin signaling and glucose uptake, GLUT4 translocation, and actin-based cytoskeleton structure. Exposure of cardiomyocytes to esculetin or NDGA, two structurally different LO inhibitors, induced a complete inhibition of insulin-stimulated glucose uptake, whereas control cells showed a threefold stimulation by insulin. Addition of 12(S)-HETE rendered the NDGA-treated cells insulin-sensitive. Early insulin signaling was not changed in cells exposed to LO inhibitors. Cell surface biotinylation of control cells showed a twofold increase of GLUT4 at the cell surface after insulin stimulation. In contrast, the LO inhibitors induced a complete inhibition of insulin-stimulated GLUT4 translocation. Labeling of the F-actin cytoskeleton revealed a prominent disassembly of actin fibers in cells exposed to the LO inhibitors. In conclusion, we show here that products of the LO reaction participate in the organization of the actin network in ventricular cardiomyocytes. Inhibition of LO blocks GLUT4 translocation without affecting insulin signaling events. These data suggest that products of the LO reaction participate in the regulation of glucose transport by contribution to a rearrangement of actin cytoskeletal elements.

Animals↗

PubMed1999

Mechanisms of internalization of Staphylococcus aureus by cultured human osteoblasts.

Staphylococcus aureus is an important bone pathogen, and evidence shows that this organism is internalized by chick osteoblasts. Here we report that S. aureus is internalized by human osteoblasts. Internalization was inhibited by monodansylcadaverine and cytochalasin D and to a lesser extent by ouabain, monensin, colchicine, and nocodazole. We propose that internalization occurs via a receptor-mediated pathway, requiring the participation of cytoskeletal elements, principally actin.

Actins↗

PubMed1988

Upstream regulatory region for inducible expression of the chicken skeletal myosin alkali light-chain gene.

The expression of the fast type of myosin alkali light chain 1 is induced during the differentiation of muscle cells. To study the mechanism of its gene regulation, we joined the sequence of the 5'-flanking and upstream region of the chicken myosin alkali light-chain gene to the structural gene for chloramphenicol acetyltransferase (CAT). The fusion gene was introduced either into quail myoblasts transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsNY68) or into chicken myoblasts, and the transiently expressed CAT activity was assayed after the differentiation of the myoblasts. From the experiments with the external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in response to the cell differentiation was found to be localized at 2 kilobases upstream of the transcription initiation site. This region of 160 nucleotides contained two pairs of short sequences worthy of note, a direct repeat of 12 nucleotides, and an inverted repeat of 8 nucleotides. The nucleotide sequences of the 5'-flanking sequence up to nucleotide -3381 were determined and compared with those of the upstream activating elements of actin genes.

Animals↗

PubMed1995

Two proximal CArG elements regulate SM alpha-actin promoter, a genetic marker of activated phenotype of mesangial cells.

Mesangial cells express smooth muscle alpha-actin (SM alpha-actin) in response to glomerular injury in vivo, and SM alpha-actin gene expression serves as a genetic marker characterizing the activated phenotype of mesangial cells. We used a molecular genetic approach to analyze the SM alpha-actin promoter and evaluate transcriptional mechanisms that might direct the genetic switch of mesangial cells to the activated phenotype. The sequence spanning -894 to +1 of the SM alpha-actin promoter directed high levels of transcription that were attenuated in serum-restricted cells and upregulated upon treatment with serum or endothelin-1. Deletional analysis revealed a core promoter fragment, from positions -122 to +1, that was necessary and sufficient for transcription. This core activity was modulated by upstream sequences between -670 and -122. The 122-bp core promoter contains two highly conserved CArG box motifs (designated CB1 and CB2), and introduction of deletion mutations of either CB1 or CB2 reduced transcription in mesangial cells to near basal levels. Further analysis revealed that CB1 and CB2 acted synergistically when subcloned upstream of a heterologous, minimal thymidine kinase promoter. CB2 alone was sufficient to confer serum inducibility to a heterologous promoter, but both CB2 and CB1 were required for maximal levels of serum-induced transcription. Collectively, these results demonstrate that CB1 and CB2 cooperate to mediate serum-induced activation of the SM alpha-actin promoter in mesangial cells.

Actins↗

PubMed2005

Interleukin-1beta induced vascular permeability is dependent on induction of endothelial tissue factor (TF) activity.

IL-1beta is a pleotropic cytokine that may mediate increased procoagulant activity and permeability in endothelial tissue during inflammatory conditions. The procoagulant effects of IL-1beta are mediated through induction of tissue factor (TF) but its alterations on vascular permeability are not well characterized. We found that IL-1beta induced a rapid and dose-dependent increase in TF activity in human umbilical vein endothelial cells (ECs) under routine culture conditions. However, IL-1beta caused a rapid and marked increase in permeability across confluent EC monolayers using a two-compartment in vitro model only in the presence of factor VIII-deficient plasma that was completely abrogated by neutralizing anti-TF antibody pre-treatment. In vitro permeability was associated with loss of EC surface expression of VE-cadherin and contraction of F-actin cytoskeletal elements that resulted in EC intercellular gap formation. These data demonstrate that IL-1beta induces marked changes in permeability across activated endothelium via a TF dependent mechanism and suggest that modulation of TF activity may represent a strategy to treat various acute and chronic inflammatory conditions mediated by this cytokine.

Journal Article↗

PubMed1988

Dynamics of membrane skeleton in fused red cell membranes.

The dynamic nature of the red cell membrane cytoskeleton was examined by the method of fluorescence redistribution after fusion (FRAF). Ghosts, labelled at the interior membrane surface, and intact cells, labelled at the outer surface with a different fluorophore, were fused and the passage of the fluorescent label into the unlabelled part of the membrane was followed in the fluorescence microscope. To achieve specific labelling of a cytoskeletal component only, a fluorescent analogue of phalloidin was used to label the actin. It was shown that there was no dissociation of the phalloidin during the time of the experiment, and no exchange of phalloidin between labelled and unlabelled actin in unsealed mixtures of ghosts. In the fused membrane pairs at 37 degrees C the phalloidin-linked fluorescence diffused in the plane of the membrane and became distributed through the membrane envelope in 1-2 h. It was shown by gel electrophoretic analysis that no detectable loss of or damage to membrane proteins resulted from the fusion process. It is concluded that, at least in membrane pairs treated with fusogen, exchange of actin or actin-containing elements occurs within the cytoskeletal network.

Cells, Cultured↗

PubMedDate not supplied

Sclerosing stromal tumor of the ovary: an ultrastructural and immunohistochemical analysis with histogenetic considerations.

Sclerosing stromal tumors are rare, benign ovarian neoplasms of unknown etiology and histogenesis. Three sclerosing stromal tumors were evaluated by immunohistochemistry and electron microscopy and were compared to two thecomas and nonneoplastic ovarian mesenchymal tissue. The sclerosing stromal tumors and thecomas were positive for muscle-specific actin; immunoreactivity was intense in the cellular areas of the sclerosing stromal tumors and focal in the thecomas. This antigen was expressed in nonneoplastic stroma predominantly in a perifollicular (theca externa) distribution. Two sclerosing stromal tumors and both thecomas were vimentin positive. Desmin was present in nonvascular cells in one of each tumor type. Expression of vimentin diffusely and of desmin focally was present in nonneoplastic cortical stroma and surrounding follicles. All specimens were nonreactive for cytokeratin. Electron microscopy supported differentiation toward smooth muscle in the sclerosing stromal tumors but not in the thecomas. Such differentiation included aggregates of cytoplasmic filaments with interspersed dense bodies, pinocytotic vesicles, and basal lamina. Delicate, long processes interconnected cells, often with primitive junctions, in the hypocellular foci. Cytoplasmic lipid, which was present in the thecomas, was not well developed in the sclerosing stromal tumors. It is proposed that a population of muscle-specific actin-positive elements exists in the theca externa--the perifollicular myoid stromal cell--and that sclerosing stromal tumors may originate from them. Sclerosing stromal tumors and thecomas share many antigenic determinants and morphologic features and thus are probably closely related entities.

Female↗

PubMed1999

Molecular motors and their role in pigmentation.

Skin pigmentation is orchestrated through a series of complementary processes. After migration of melanoblasts out of the neural crest to epidermis and hair follicle, these cells mature into melanocytes. Differentiated melanocytes produce melanin in specialized organelles, the melanosomes. Moreover, the cytoplasm of melanocytes branches into extensions, the dendrites. Via the tips of these dendrites they donate their mature melanosomes to the keratinocytes resulting in skin pigmentation. Thus, one essential part of the process of pigmentation is the translocation of melanosomes from their site of origin in the perinuclear cytoplasm towards the dendrite tips. Motor proteins are molecules which use the energy derived from ATP hydrolysis to move along cytoskeletal elements, either actin filaments or microtubules, to transport their cargo, which can be organelles, vesicles or chromosomes. This review describes the different classes of microtubule-based and actin-based motor proteins with their characteristics and functional importance in cell biology and organelle transport. Some of them will be highlighted and several recent studies in mammalian pigment cells indicating their role in pigment granule transport will be discussed. As a result of these data and previous suggestions, a model will be proposed for the possible cooperation of both systems in melanosome movement.

Actin Cytoskeleton↗

PubMed2002

Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor-dependent mechanism: relationship between the procoagulant and permeability effects of TNF.

Tumor necrosis factor (TNF) has marked effects on permeability and procoagulant activity on tumor-associated neovasculature when used in isolation perfusion, the latter effect primarily mediated via induction of cell surface expression of tissue factor (TF) on endothelial tissue. However, the cellular events that result in rapid alterations in endothelial cell (EC) permeability after intravascular TNF administration in isolation perfusion are not well characterized. We demonstrate that short exposure intervals to TNF induces TF expression on ECs but has no effect on permeability as assessed by flux of Evans blue-bound albumin across confluent EC monolayers using a 2-compartment model under basal culture conditions. However, a rapid and significant increase in EC permeability occurred with TNF in the presence of factor VIII-deficient plasma. Permeability was induced only with luminal versus abluminal TNF exposure and was blocked by antithrombin III, TF pathway inhibitor, or anti-TF antibody cotreatment. These data indicate that EC surface expression of TF and extrinsic clotting factors are critical in augmenting capillary leak following intravascular TNF administration. Alterations in permeability were associated with intercellular gap formation at sites of down-regulation of vascular endothelial (VE)-cadherin expression, the primary endothelial intercellular adhesion molecule, and intracellular contraction and alignment of F-actin cytoskeletal elements. Rapid induction of TF by TNF may be the primary EC response that results in alterations in permeability and procoagulant activity observed following intravascular TNF administration in isolation perfusion.

Antibodies, Monoclonal↗

PubMed1989

Immunohistochemical study of a case of malignant müllerian mixed tumor in comparison with the activity of normal uterine tissue.

A case of malignant Müllerian mixed tumor of the uterus, exhibiting a histology of heterologous osteosarcomatous differentiation, is presented. Special emphasis is placed on the characteristic immunohistochemical reactivity of the tumor tissue in comparison with that of normal uterine tissue in the proliferative phase. Keratin, cytokeratin, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (HCG) were found only in the carcinomatous element. However, neuron specific enolase (NSE), S-100 protein (S 100) and vimentin were identified in almost all tumor tissue elements. Desmin and actin were not stained in any elements. Myoglobin was only detected weakly in the squamous carcinomatous element. Undifferentiated cell element, composed of small, round, spindle, or polygonal cells showed positive reactions to NSE and S 100, but not to any other antibodies. As compared to the reactivities of the normal proliferative endometrium, the glandular epithelial cells were positive with NSE, S 100, vimentin and CEA, but the stromal cells were only positive with vimentin. Such a multitudinous and concomitant expression of antigenicity to the different tumor elements indicates a close relationship to its mesodermal Müllerian origin, and NSE, S 100 and vimentin might be most adequate indicators of these types of tumors.

Adenocarcinoma↗

PubMed1983

Epiretinal membranes in vitro.

Monolayers of cells were grown from epiretinal membranes using standard tissue culture techniques. The growing cells were filmed by time-lapse cinephotomicrography to record their locomotory characteristics and cell-to-cell interactions. The established layers of cells were examined in the scanning electron microscope. In addition, the cultured cells were studied immunohistochemically to identify specific cell types and the distribution of contractile elements (e.g. actin). The locomotory patterns, the topography and the immunohistochemistry of cells from epiretinal membranes were compared to the movement, appearance and staining characteristics of a wide range of normal cells in culture. From the growth characteristics in tissue culture, early membranes appeared more hypercellular than longer standing membranes. Cell outgrowth was established more quickly and was more widespread in the young epiretinal membranes than in older, more fibrous, specimens. The locomotory and immunohistochemical studies demonstrated that a wide variety of cell types may contribute to epiretinal membranes. A remarkably wide range of cell forms were observed in cultures of individual membranes. Epithelial and glial cells were found in many of the monolayers. However, they did not constitute the majority of cells in the cultures. The predominating cell types had the morphological and locomotory characteristics of fibroblasts or macrophages.

Animals↗

PubMed2003

A transforming growth factor-beta control element required for SM alpha-actin expression in vivo also partially mediates GKLF-dependent transcriptional repression.

We previously demonstrated that a conserved transforming growth factor-beta control element (TCE) within the 5'-region of the smooth muscle cell (SMC) differentiation marker gene SM alpha-actin could mediate both transcriptional activation and repression in cultured SMCs through interaction with members of the zinc finger Kruppel-like transcription factor (KLF) family. The aims of the present studies were to: 1) determine the role of the SM alpha-actin TCE in vivo through mutagenesis studies in transgenic mice and 2) further characterize the possible role and mechanisms by which the TCE-binding factor GKLF/KLF4 induces repression of SMC marker genes in various SMC model systems in vitro. Our results showed that the TCE was required for SM alpha-actin promoter activity in transgenic mice in vivo. Results of transient transfection studies showed that GKLF-induced repression of a SM alpha-actin promoter/luciferase reporter gene partially depended on the TCE. Furthermore, a GKLF overexpressing adenovirus inhibited whereas GKLF morpholino antisense oligos increased expression of endogenous SMC marker genes. Results of chromatin immunoprecipitation assays showed GKLF binding to TCE containing regions of various SMC marker gene promoters within intact chromatin. Finally, results of co-transfection studies showed that overexpression of IKLF/KLF5 reversed GKLF-dependent repression thus supporting a model of reciprocal activation-repression of SMC gene expression by different members of the KLF gene family.

Actins↗

PubMed2002

RNA trafficking and stabilization elements associate with multiple brain proteins.

Two of the best understood somatic cell mRNA cytoplasmic trafficking elements are those governing localization of beta-actin and myelin basic protein mRNAs. These cis-acting elements bind the trans-acting factors fibroblast ZBP-1 and hnRNP A2, respectively. It is not known whether these elements fulfil other roles in mRNA metabolism. To address this question we have used Edman sequencing and western blotting to identify six rat brain proteins that bind the beta-actin element (zipcode). All are known RNA-binding proteins and differ from ZBP-1. Comparison with proteins that bind the hnRNP A2 and AU-rich response elements, A2RE/A2RE11 and AURE, showed that AURE and zipcode bind a similar set of proteins that does not overlap with those that bind A2RE11. The zipcode-binding protein, KSRP, and hnRNP A2 were selected for further study and were shown by confocal immunofluorescence microscopy to have similar distributions in the central nervous system, but they were found in largely separate locations in cell nuclei. In the cytoplasm of cultured oligodendrocytes they were segregated into separate populations of cytoplasmic granules. We conclude that not only may there be families of trans-acting factors for the same cis-acting element, which are presumably required at different stages of mRNA processing and metabolism, but independent factors may also target different and multiple RNAs in the same cell.

Actins↗

PubMed1996

Distal cis-acting elements restrict expression of the CyIIIb actin gene in the aboral ectoderm of the sea urchin embryo.

The distal region of the S. purpuratus actin CyIIIb gene, between -400 and -1400 nucleotides, contains at least three distinct cis-acting elements (C1R, C1L and E1) which are necessary for correct expression of fusion reporter genes in transgenic sea urchin embryos. The contribution of these elements in the temporal and spatial regulation of the gene was analyzed by single and double site-directed mutagenesis in fusion constructs which carry the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter. Following microinjection of the transgenes in sea urchin embryos, the activity of the mutants was compared to the wild type in time and space by measuring CAT activity at the blastula and pluteus embryonic stages and by in situ hybridization to the CAT mRNA at pluteus stage. Our results indicate that E1 is involved in the temporal regulation of CyIIIb and that all three elements are necessary and sufficient to confer aboral (dorsal) ectoderm specificity to the proximal promoter. This is achieved by suppressing the promoter's activity in all other tissues by the cooperative interaction of the cis-acting elements. The C1R element, binding site of the nuclear receptors SpCOUP-TF and SpSHR2, is by itself sufficient to restrict expression in the ectoderm, whereas the aboral ectoderm restricted expression requires in addition the presence of both C1L and E1. It is therefore evident, that the actin CyIIIb gene is exclusively expressed in the aboral ectoderm by a combinatorial repression in all other cell lineages of the developing embryo.

Actins↗

PubMed1993

Upstream elements involved in the embryonic regulation of the sea urchin CyIIIb actin gene: temporal and spatial specific interactions at a single cis-acting element.

The sea urchin Strongylocentrotus purpuratus CyIIIb actin gene codes for a cytoskeletal type actin and is activated in the early embryo specifically in the cells destined to become the aboral ectoderm. Deletion constructs of its upstream region fused to the bacterial chloramphenicol acetyltransferase (CAT) gene were expressed in developing embryos following microinjection into eggs. These studies revealed the segments of the upstream region which are necessary for embryonic expression. We mapped the protein:DNA interaction sites in this upstream region by DNase I footprinting. At least five binding sites were identified in the 2173 nucleotide flanking sequence, which seem to include all the necessary elements for quantitative expression. One of these elements (E1) showed different patterns of association in vitro with nuclear proteins isolated from different stages in development. These different interactions are separated when nuclear extracts from ectodermal or endodermal-mesodermal tissues were used. When S. purpuratus eggs were injected with a CAT fusion construct mutated at this site, a decrease in CAT activity was observed only in late stage embryos (plutei), whereas early stage embryos (blastulae) exhibited wild-type CAT activity. These results suggest that the E1 element is involved in the temporal regulation of the CyIIIb gene.

Actins↗

PubMed1987

Serotonin induced actin polymerization and association with cytoskeletal elements in cultured bovine aortic endothelium.

Serotonin, (5-hydroxytryptamine, 5-HT) binds to cultured endothelial cell stress fibers as identified by fluorescence microscopy and in vitro induces actin polymerization as measured by DNase 1 inhibition and differential centrifugation; the structural change in actin in the presence of 5-HT resembles actin paracrystals as visualized by negative staining under electron microscopy. These observations indicate that 5-HT, which is taken up by endothelium by a non-mediated diffusion, may interact directly with actin to affect cytoskeletal changes.

Actins↗