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PubMedDate not supplied

Actin microfilaments dynamics in African green monkey renal cell line (Vero) during cultivation.

The structure of the actinic cytoskeleton in Vero cells (African green monkey kidney cells) during monolayer formation was studied. By immunofluorescence after phalloidin staining, actin was visualized in cells 15 min, 2, 4, 24 and 48 hours after cultivation. The evolution in time of cells revealed the progressive organization of actin molecules from diffuse granular forms to filamentous forms with characteristic patterns. The evolution of actinic cytoskeletal elements was closely correlated with the evolution of the cell shape and the extension of intercellular contact surfaces.

Actin Cytoskeleton↗
PubMed2001

Regulated expression of human filaggrin in keratinocytes results in cytoskeletal disruption, loss of cell-cell adhesion, and cell cycle arrest.

Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.

Adherens Junctions↗
PubMed1997

Identification of two regions in the N-terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes.

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N-terminus of ActA (residues 1-234) to the omega fragment of beta-galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region T, amino acids 117-121) is critical for filament elongation, as shown by the absence of actin tail in a 117-121 deletion mutant or when motility assays are performed in the presence of anti-region T antibodies. The second region (region C, amino acids 21-97), is more specifically involved in maintenance of the continuity of the process, probably by F-actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21-97) leading to 'discontinuous' actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33-74 can interact with F-actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N-terminal domain of ActA.

Actins↗
PubMed1999

Modulation of volume-sensitive Cl - channels and cell volume by actin filaments and microtubules in human cervical cancer HT-3 cells.

Hypotonicity activates volume-sensitive Cl- currents, which are implicated in the regulatory volume decrease (RVD) responses and transport of taurine in human cervical cancer HT-3 cells. In this study, the role of cytoskeleton in the regulation of volume-sensitive Cl- channels and RVD responses in HT-3 cells was studied. Cells were incubated with various compounds, which depolymerized or polymerized cytoskeletal elements, i.e. actin filaments and microtubules. The hypotonicity-induced changes in Cl- conductance and in cell volume were measured by whole-cell voltage clamping and cell size monitoring, respectively. Our results show that in HT-3 cells hypotonicity activated an outward rectified Cl- current that was abrogated by Cl- channel blockers. Cytochalasin B, an actin-depolymerizing compound, induced a substantial increase in Cl- conductance under isotonic condition and potentiated the expression of Cl- currents in hypotonic stress. Phorbol 12-myristate 13-acetate (PMA) significantly inhibited the cytochalasin B-induced activation of Cl- conductance under isotonic condition. On the other hand, treatment with cytochalasin B significantly prolonged the RVD responses. Phalloidin, a stabilizer of actin polymerization, did not change the basal currents under isotonic condition, but completely abolished the increase in whole-cell Cl- conductance elicited by hypotonicity and retarded the cell volume recovery. Colchicine, a microtubule-assembly inhibitor, had no effect on either basal Cl- conductance or volume-sensitive Cl- current and was unable to inhibit the RVD responses. Taxol, a microtubule-stabilizing compound, did not alter the basal Cl- conductance, but inhibited the activation of volume-sensitive Cl- channels as well as the process of RVD in a dose-dependent manner. These data support the notion that functional integrity of actin filaments and microtubules plays critical roles in maintaining the RVD responses and activation of Cl- channels in human cervical cancer HT-3 cells.

Actins↗
PubMed1998

Three-dimensional matrix primes mesangial cells to down-regulation of alpha-smooth muscle actin via deactivation of CArG box elements.

Prolonged culture of mesangial cells forms multifocal nodular structures, termed "hillocks," composed of cells and extracellular matrix (ECM), which may mimic the situation in the glomerular mesangium. Mesangial cells incorporated in hillocks show repressed expression of alpha-smooth muscle actin, a marker of mesangial cell activation/dedifferentiation. The aim of this study is to elucidate molecular mechanisms involved in this phenomenon, focusing on the activity of CArG box elements located in 5'-flanking region of the alpha-smooth muscle actin gene. Reporter mesangial cells were created to monitor the activity of CArG elements. These clones expressed beta-galactosidase gene (lacZ) under the control of CArG boxes. Within the hillocks, reporter cells showed repressed expression of lacZ as well as alpha-smooth muscle actin compared to the cells in two-dimensional cultures. Consistent with this result, the reporter cells embedded in collagen gel exhibited down-regulation of lacZ and alpha-smooth muscle actin transcripts. Deactivation of CArG box elements by transfection with either a dominant negative mutant of serum response factor or a dominant negative form of ternary complex factor Elk-1 led to depressed expression of alpha-smooth muscle actin gene. These data suggested that three-dimensional ECM primes mesangial cells to down-regulation of alpha-smooth muscle actin via deactivation of CArG box elements.

Actins↗
PubMed1989

DNA bending is induced by a transcription factor that interacts with the human c-FOS and alpha-actin promoters.

Conserved sequence elements in the human cardiac and skeletal alpha-actin promoters that contain the CC(A + T-rich)6GG motif have been shown to regulate transcription of these genes. A similar sequence is found in the serum response element of the human c-FOS gene. In this study, we demonstrate that indistinguishable proteins bind to each of five CC(A + T-rich)6GG elements examined in the human cardiac and skeletal alpha-actin promoters and the c-FOS serum response element. Using electrophoretic techniques, we show that these factors induce a stable bend in the DNA upon binding, and the bend center is shown to coincide with the CC(A + T-rich)6GG element. In addition, the ability to bend DNA is retained by a small proteolytic fragment of the protein, suggesting that the DNA-binding domain of the protein is resistant to proteases and is sufficient to bend DNA.

Actins↗
PubMed1999

Actin-rich desmoplastic malignant melanoma: report of three cases.

We report three cases of desmoplastic malignant melanoma (DMM) rich in smooth muscle actin. They occurred in two men (Cases 1 and 3) and in one woman (Case 2). Cases 1 and 2 were recurrent lesions from common melanomas excised, respectively, 3 and 1 years previously. In Case 3, DMM was associated with lentigo maligna at the time of presentation. Morphologically, DMMs were composed of spindle neoplastic cells organized in haphazardly orientated long fascicles separated by collagen bundles. Perineural invasion was present and mitotic activity was prominent in all cases. The neoplastic spindle cells were intensely positive with S100 protein and smooth muscle actin antisera and negative with HMB45 and Melan-A (Mart-1) antibodies. Double staining for smooth muscle actin and S100 protein revealed no definite coexpression of the two antigens. Follow-up was available for patients 1 and 2 who had local recurrences and are still alive. It is possible that actin rich elements differentiate toward mesenchymal elements, paralleling the phenotypic changes seen in sarcomatoid carcinomas. Therefore, multidirectional differentiation may explain the mesenchymal (sarcomatoid) differentiation of neoplastic melanocytes and may be responsible for the different biologic behavior of DMMs, which is closer to mesenchymal tumors than to conventional melanomas.

Actins↗
PubMedDate not supplied

The cytoskeleton as a subcellular target of the antineoplastic drug lonidamine.

Lonidamine (LND), a dichlorinated derivative of indazole-3-carboxylic acid, has proved to exert a powerful antiproliferative effect and to impair the energy metabolism of normal and neoplastic cells. A target effect of the drug on the cell membrane structure was hypothesized. Thus, in order to elucidate better the mechanism of action of LND, the drug effects on the cell surface as well as on main cytoskeletal elements, i.e. actin microfilaments, microtubules and intermediate filaments, were investigated. In particular, an immunocytochemical and ultrastructural study was performed using two different cell lines: epithelial squamous carcinoma (A431) and melanoma (M14) cells. Treatment with 0.8 mM LND for 8 hr induced a remarkable rearrangement of the F-actin molecules with the disappearance of the stress fibers. As far as microtubules are concerned, formation of perinuclear patches of tubulin were detected after LND treatment. Intermediate filaments appeared to be differently affected by LND in the two cell types. Such changes were detected as an early phenomenon and the extent of the effects observed was positively related to the cell surface alterations and to the loss of cell viability, suggesting that the cytoskeletal elements might represent an additional target in the mechanisms of cytotoxic action of LND.

Antineoplastic Agents↗
PubMed2000

Evolution of cis-acting elements in 5' flanking regions of vertebrate actin genes.

Regulation of the vertebrate actin multigene family involves the recognition of various regulatory sequences (cis-acting elements) that specify the distinct tissue type and developmental program of expression for each actin paralogue, which implies that the distribution of cis-acting elements may be unique for each paralogue gene. To elucidate the evolution of these unique distribution patterns, we improved a method to scan for cis-acting elements in the 5' flanking regulatory region of genes and used it to analyze five cis-acting elements (SRE, MyoD binding site, Elk-1 binding site, positive and negative YY1 binding sites) of six actin paralogue genes (beta and gamma cytoplasmic actins, alpha and gamma smooth muscle actins, and alpha skeletal and alpha cardiac actins) among various vertebrates. It was shown that although an element(s) may exist in all paralogue genes of the same species, its numbers, compositions, and distribution patterns or even sequences vary remarkably among paralogues, which contributes to their different tissue- and developmental-specific expression. However, each pair of coexpressed paralogues has some certain similarity in distribution patterns. Furthermore, among various orthologues of actin genes derived from diverse vertebrates, the sequences, numbers, and distribution patterns of these cis-acting elements are highly conserved or even identical in the long run of phylogeny of vertebrates. Taken together, the results described above strongly indicate that not only the structures of actins but also their expression patterns are essential in both the phylogeny and the physiology of vertebrates. The distribution patterns of cis-acting elements of various actin genes can be regarded as indicators of both horizontal (paralogues) and vertical (orthologous) evolution of actins.

Actins↗
PubMed2003

Effects of chilling on structural aspects of early preantral mouse follicles.

Chilling injury is one of the major limiting factors for achieving optimal cryopreservation of gametes. This study aimed to determine potential chilling-induced damage on several structural aspects of early preantral mouse follicles. Mechanically isolated intact early preantral follicles (type 3b-4) were exposed to 0 degrees C for 1, 5, 10, or 30 min. Control and chilled follicles were analyzed by confocal microscopy after staining for tubulin, F-actin, and chromatin, and by electron microscopy. Chilling for only 1 min was sufficient to cause depolymerization of microtubules in the oocyte and the surrounding granulosa cell layer as evidenced by a substantial decrease in fluorescence intensity after antitubulin labeling. Cooling for longer periods caused alterations in microtubule organization in the follicle-enclosed oocyte. These alterations included the loss of interphase microtubules, concomitant with the formation of perinuclear or cortical microtubule asters and sometimes a complete disappearance of microtubules. The extent of microtubule modification was related to the time of chilling, but was fully reversible after rewarming follicles at 37 degrees C for 1 h. Chilling had only minor effects on the actin-containing elements located predominantly in the oocyte cortex and the transzonal projections. Ultrastructural analysis confirmed that oocyte-somatic cell interactions were present. There was no influence on the chromatin configuration within the follicle-enclosed oocyte. These results indicate that mouse follicles are relatively tolerant to direct chilling injury and, as a consequence, are able to withstand the cooling-warming steps during conventional cryopreservation procedures.

Actin Cytoskeleton↗
PubMed2002

WAVE3, an actin-polymerization gene, is truncated and inactivated as a result of a constitutional t(1;13)(q21;q12) chromosome translocation in a patient with ganglioneuroblastoma.

Neuroblastoma (Nb) is a malignancy of the sympathetic nervous system which affects children in their first decade. It is the most common extra-cranial solid tumor in children with an incidence of approximately 1 in 8-10 000 live births annually and accounts for approximately 10% of all children's cancers. Ganglioneuroblastoma is a relatively benign form of Nb and consists of a mixture of fibrils, mature and maturing ganglion cells, as well as undifferentiated neuroblasts. During routine cytogenetic analysis of patients with different manifestations of neuroblastoma we have identified one patient with ganglioneuroblastoma that carries an apparently balanced t(1:13)(q21:q12) reciprocal translocation. Positional cloning of the translocation breakpoint on chromosome 13 resulted in the mapping of the breakpoint between coding exon 2 and exon 3 of WAVE3, a member of WASP gene family. Although the breakpoint region on chromosome 1 was localized to within 2 kb of genomic sequence, no gene was found to be interrupted on this chromosome. The WAVE3 transcript is mainly expressed in the nervous system and, like all the members of the WASP gene family, WAVE3 is a key element in actin polymerization and cytoskeleton organization. WAVE3, therefore, is important for cell differentiation and motility and its expression is lost in a number of low grade and stage 4S tumors. From analysis of its expression pattern and function, WAVE3 is a candidate tumor suppressor gene, at least in some forms of neuroblastoma.

Actins↗
PubMed1995

Topographic compensation: guidance and directed locomotion of fibroblasts on grooved micromachined substrata in the absence of microtubules.

Fibroblasts cultured on grooved substrata align themselves and migrate in the direction of the grooves, a phenomenon called contact guidance. Microtubules have been deemed important for cell polarization, directed locomotion, and contact guidance. Because microtubules were the first cytoskeletal element to align with the grooves when fibroblasts spread on grooved substrata, we investigated the consequences of eliminating the influence of microtubules by seeding fibroblasts onto smooth and grooved micromachined substrata in the presence of colcemid. Fibroblasts were examined by time-lapse cinematography and epifluorescence or confocal microscopy to determine cell shape and orientation and the distribution of cytoskeletal or associated elements including actin filaments, vinculin, intermediate filaments, microtubules, and kinesin. As expected, cells spreading on smooth surfaces in the presence of colcemid did not polarize or locomote. Surprisingly however, by 24 hours, cells spread on grooves in the presence of colcemid were morphologically indistinguishable from controls spread on grooves. Both groups were aligned and polarized with the direction of the grooves and demonstrated directional locomotion along the grooves. In the absence of microtubules, kinesin localized to some of the aligned stress fibers and to leading edges of cells spreading on grooves. The grooved substratum compensated for the microtubule deficiency by organizing and maintaining an aligned actin filament framework. Thus, microtubules are not required to establish or maintain stable, polarized cell shapes or directed locomotion, provided an alternate oriented cytoskeletal component is available.

Actins↗
PubMed1992

Distribution of the surface antigen HAM-4 and cytoskeleton during reformation of bile-canalicular structures in rat primary cultured hepatocytes.

The distribution of rat bile-canalicular surface antigen (HAM-4 antigen) and cytoskeletal elements (microtubules, actin filaments, and cytokeratin filaments) was examined during the reformation of bile-canalicular structures (BC-structures) in primary cultures of dissociated hepatocytes obtained following collagenase perfusion. HAM-4 antigen, which initially dispersed after cell dissociation, became focused into regions of cell-to-cell contact even before formation of BC-structures. Typical bile-canalicular microvilli also appeared in these regions before the intercellular spaces were completely closed. Finally, after in vitro reformation of BC, HAM-4 antigen was localized specifically at the BC-surface. The process of BC-reformation and the intracellular organization of actin and cytokeratin filaments were not significantly affected by microtubule inhibitors (nocodazole, colcemid, and colchicine). However, the localization of HAM-4 antigen molecules at the surface of BC was disrupted by these inhibitors, suggesting that the distribution of HAM-4 antigen, which represents a marker for the reconstruction of surface polarity, is dependent on microtubule function.

Animals↗
PubMed1995

Nuclear scaffold-associated protease: in situ nuclear localization and effects of a protease inhibitor on growth and morphology of a ras-transformed hepatocyte cell line.

We have previously identified a multicatalytic protease (MCP) activity associated with the nuclear scaffold (NS) in hepatocytes and fibroblasts. When we used the chloromethylketone protease inhibitor AAPFcmk, which is targeted to chymotrypsinlike protease activity, we observed a dramatic inhibition of transformation of fibroblasts, with effects that were relatively selective for the NS fraction. Here, we undertook experiments to determine the effects of AAPFcmk on Simian Virus 40-immortalized CWSV1 cells compared with a ras-transformed hepatocyte cell line (NR4) derived from CWSV1. We used biotinAAPFcmk and fluorescent reagents to demonstrate a nuclear chymotrypsinlike protease activity, which is most prominent at the nuclear envelope. The ras-transformed NR4 cells were highly susceptible to growth inhibition in a dose-dependent manner, showing 85% growth inhibition at 50 mumol/LAAPFcmk. In contrast, the immortalized CWSV1 cells were not sensitive at the concentrations (10 to 50 mumol/L) of AAPFcmk tested. In subcellular fractionation studies, the inhibitory effects of AAPFcmk were confined to the NS fraction. The AAPFcmk-induced growth inhibition was accompanied by marked morphological changes in ras-transformed cells, without evidence of overt toxicity. No change in DNA content was observed, but a marked increase in organization of actin cytoskeletal elements was seen. These results suggest that a protease activity associated with the nuclear scaffold has important functions in controlling cytoskeletal filament organization and cell replication.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Intermediate filaments and their associated proteins: multiple dynamic personalities.

A fusion of mouse and human genetics has now proven that intermediate filaments form a flexible scaffold essential for structuring cytoplasm in a variety of cell contexts. In some cases, the formation of this scaffold is achieved through a newly identified family of intermediate-filament-associated proteins that form cross-bridges between intermediate filaments and other cytoskeletal elements, including actin and microtubules.

Animals↗
PubMed1992

Effects of tumour cells on angiogenesis and vasoconstrictor responses in sponge implants in mice.

The effects of tumour cells (Colon 26) on the development and response of new blood vessels to different vasoconstrictors (platelet activating factor; PAF, endothelin-1, angiotensin II, adrenalin and 5-hydroxytryptamine) have been investigated. Sponge implants in mice were used to host tumour cells while washout of 133Xe was employed to assess local blood flow in the implanted sponges. By 14 days after implantation the response of vessels in tumour-bearing implants to the various vasoconstrictors generally was decreased compared to that obtained in control sponge implants or adjacent normal skin. Thus at this time point the t1/2 for 133Xe washout from control sponges treated with adrenalin (0.5 micrograms) was 30 +/- 4 min whereas in tumour-bearing sponges it was 5 +/- 1 min. This decreased sensitivity in tumour vessels was probably not due to a complete lack of contractile elements since actin was demonstrated by immunohistochemistry around blood vessels in both types of implant. The results of the present study have shown that the pharmacological responses of blood vessels in a growing tumour, Colon 26, differed from the responses of vessels of a similar age in non-neoplastic tissue. These results appear to suggest that the different angiogenic stimuli released from tumour tissue may markedly influence pharmacological reactivity of newly formed blood vessels.

Adenocarcinoma↗
PubMed1995

Heparin-induced endothelial cell cytoskeletal reorganization: a potential mechanism for vascular relaxation.

We have previously shown that heparin given subcutaneously on a daily basis lowers blood pressure in hypertensive rat models, and that this blood pressure lowering effect is endothelium-dependent. The present study describes the effects of heparin on endothelial cell (EC) apical surface structures and cytoskeletal elements, namely, actin and vimentin as well as EC proliferative activity. The EC line (CRL 1998) was cultured, treated with different concentrations of heparin (0, 50, 100, 500 U/ml) for 4, 24 or 48 hours, and fixed for scanning electron microscopy (SEM), and immunofluorescence microscopy (IFM) studies. Enzyme-linked immunosorbent assays (ELISA) and flow cytometric analysis were performed on EC monolayers treated with different concentrations of heparin for quantitative detection of actin and vimentin. By SEM study the cell surface showed generalized smoothing as a result of blunting of surface microvilli with increasing time of exposure and dosage of heparin. By IFM study, the detectable actin signal within ECs became progressively reduced in both its cellular distribution and the apparent number of cells that remained reactive. By 48 hr/500 U heparin, the actin signal was almost undetectable. Vimentin showed a moderate reduction in the cellular distribution of labeling. Quantitatively, actin was significantly reduced after the 24 hour treatment with a higher dose of heparin (500 U/ml), from a baseline optical density (OD) of 1.12 +/- 0.060 to 0.866 +/- 0.008 (P < 0.0027). After 48 hours of treatment at both 100 U/ml and 500 U/ml heparin, actin was significantly reduced from a baseline OD of 1.347 +/- 0.063 to 1.090 +/- 0.039 (P < 0.0039) and 0.844 +/- 0.074 (P < 0.008), respectively. However, vimentin was significantly reduced only after 48 hours of treatment with a high dose of heparin (500 U/ml), from baseline OD 1.82 +/- 0.052 to 1.41 +/- 0.004 (P < 0.002). The flow cytometric findings were virtually identical to the ELISA data for actin and vimentin. These qualitative and quantitative changes in actin and vimentin are consistent with apparent smoothing and relaxation of the EC's apical surface. Labeling with the cell cycle marker MIB-1 (monoclonal antibody Ki-67), showed a progressive reduction in the observed intensity in heparin treated cells with substantially fewer cells being positive. After a 48 hour treatment with heparin (500 U/ml), most ECs displayed only dim labeling of the nucleolus. This finding is consistent with an antiproliferative effect. Overall, these findings are additive to our previous observations, and demonstrate that heparin causes EC cytoskeletal reorganization which is a potential mechanism for vascular relaxation.

Cytoskeleton↗