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PubMed1995

Cytoskeletal regulation of Caco-2 intestinal monolayer paracellular permeability.

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and processes that regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments, and microtubules. Cytochalasin-b (5 micrograms/ml) and colchicine (2 x 10(-5) M) at the doses used caused marked depolymerization and disruption of actin microfilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracellular permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway "re-tightening" following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercellular junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin microfilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes. This re-closure process did not require protein synthesis or microtubule-mediated shuttling process.

Actins↗

PubMed2005

The development of a long, coiled, optic nerve in the stalk-eyed fly Cyrtodiopsis whitei.

In the stalk-eyed fly Cyrtodiopsis whitei (Diopsidae; Diptera), the relatively long optic nerve develops within the tight lumen of a very short eyestalk. Axonal growth is generally considered in terms of path finding, selective fasciculation, and towing. Physical forces that are necessary for axon lengthening are generated either by the growth cone or by the growth of surrounding tissues. Therefore, it is surprising to encounter a loosely coiled nerve apparently lacking any attachments that could allow for pull, or towing, of the nerve. In this study, we used histological sections and whole-mount preparations to confirm that the optic nerve of the stalk-eyed fly indeed elongates without the external application of tension to the nerve. Secondly, we examined the distribution of cytoskeletal elements and selected proteins that may be involved in axon extension. Staining against the vesicle fusion proteins SNAP-24 and SNAP-25 consistently results in stronger staining in the rapidly extending optic nerve than in a control nerve, suggesting a possible role of these proteins in the extension process. On a gross morphological level, SNAP-24/25 as well as the cytoskeletal elements actin and tubulin are uniformly distributed throughout the lengths of the growing nerve, suggesting that nerve elongation is distributed rather than localized. Finally, we identified glia as a possible source for tension within the nerve bundle. Glia proliferate rapidly in the optic nerve but not in the control nerve. Much work continues to focus on the growth of axons in culture, but this study is one of the few that considers the dynamics of nerve bundle extension as a whole.

Actins↗

PubMed1984

Control of cell shape and locomotion by external calcium.

Dependence of locomotion of Xenopus laevis epidermal cells on calcium influx from the external medium was investigated. Inhibition of Ca2+ influx by 2 mM La3+ or 4 mM Tb3+ in the culture medium causes an immediate stop to locomotion and a loss of motion at the outer margin of the lamella; microcolliculi disappear and the entire lamella becomes flat and very thin. The cell body region enlarges by spreading into the lamella to an extent approximately coincident with the distribution of myosin. The increase in thickness of this area is the result. The cytoskeletal elements actin, alpha-actinin and myosin become homogeneously distributed throughout the cell and a great number of straight microtubules extend to the margin after 20 min in La3+-containing media. Prekeratin distribution does not change. Reduction of calcium concentration in the external medium by EGTA leads to cessation of cell locomotion. Sr2+ (1-4 mM) is also able to replace calcium for triggering locomotion. These findings point to a control of Ca2+-activated contractions of actomyosin by influx of external Ca2+. According to our model of cell locomotion [14] the contractions generate a hydrostatic pressure extending the lamella by flow of hyaloplasm towards the margin. Small swellings (microcolliculi) appearing thereby will be dislocated by a calcium-dependent sol-gel transformation in this area, which contains actin but not myosin.

Animals↗

PubMed2004

The machinery of mitochondrial fusion, division, and distribution, and emerging connections to apoptosis.

Mitochondrial undergo regulated fusion and division in many organisms and cell types, and each event is mediated by a different complex of proteins each containing at least one large GTPase. The mitochondrial fusion and division molecular machinery is in large part conserved; recent studies show a functional connection between some of these proteins and the apoptotic cascade. Mitochondria also undergo directed movement in cells, and the gene products that attach and propel mitochondria along cytoskeletal elements (actin filaments in some organisms, microtubules in others) are becoming gradually elucidated.

Journal Article↗

PubMed2005

Dystonia-causing mutant torsinA inhibits cell adhesion and neurite extension through interference with cytoskeletal dynamics.

Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms-recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA-the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.

Actins↗

PubMed1983

Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.

Actins↗

PubMed1992

Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation.

The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes. They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles. Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC. However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the Fc epsilon RI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4 degrees C or in the absence of extracellular calcium. Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events.

Actins↗

PubMed2001

Polarized cell growth in higher plants.

Pollen tubes and root hairs are highly elongated, cylindrically shaped cells whose polarized growth permits them to explore the environment for the benefit of the entire plant. Root hairs create an enormous surface area for the uptake of water and nutrients, whereas pollen tubes deliver the sperm cells to the ovule for fertilization. These cells grow exclusively at the apex and at prodigious rates (in excess of 200 nm/s for pollen tubes). Underlying this rapid growth are polarized ion gradients and fluxes, turnover of cytoskeletal elements (actin microfilaments), and exocytosis and endocytosis of membrane vesicles. Intracellular gradients of calcium and protons are spatially localized at the growing apex; inward fluxes of these ions are apically directed. These gradients and fluxes oscillate with the same frequency as the oscillations in growth rate but not with the same phase. Actin microfilaments, which together with myosin generate reverse fountain streaming, undergo rapid turnover in the apical domain, possibly being regulated by key actin-binding proteins, e.g., profilin, villin, and ADF/cofilin, in concert with the ion gradients. Exocytosis of vesicles at the apex, also dependent on the ion gradients, provides precursor material for the continuously expanding cell wall of the growing cell. Elucidation of the interactions and of the dynamics of these different components is providing unique insight into the mechanisms of polarized growth.

Actin Cytoskeleton↗

PubMed1996

SRF and TEF-1 control of chicken skeletal alpha-actin gene during slow-muscle hypertrophy.

The purpose of this study was to delineate the alpha-actin regulatory elements and transcription factors that are responsible for conferring stretch-overload responsiveness during hypertrophy of the anterior latissimus dorsi (ALD) muscle of young chickens by weighting one wing. Minimal promoter constructs were evaluated by direct injection into the ALD, which demonstrated that both serum response element 1 (SRE1) and the transcriptional enhancer factor 1 (TEF-1) elements were sufficient for increased expression during stretch overload. A mutated SRE1 prevented expression in both basal and stretched ALD muscles, whereas a mutated TEF-1 element reduced actin promoter function in both control and stretched muscles. The serum response factor (SRF)-SRE1 binding complex demonstrated faster migration in mobility shift assays from day 3-and day 6-stretched ALD nuclear extracts relative to their control. TEF-1 binding was qualitatively increased in stretched extracts at day 3 but not day 6 of stretch overload. Skeletal alpha-actin mRNA accumulated from day 3 to day 6 of stretch overload. These data demonstrate that SRE1 is necessary and sufficient for stretch-overload responsiveness from the skeletal alpha-actin promoter and that the SRF-SRE1 binding complex migrates faster in stretched nuclear extracts of hypertrophied relative to control extracts from intact ALD muscles of chickens.

Actins↗

PubMed2005

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction.

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.

Actin Cytoskeleton↗

PubMed2003

Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity.

During erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of erythroblast plasma membrane components to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about molecular mechanisms governing plasma membrane protein sorting. We chose glycophorin A (GPA) as a model integral protein to begin investigating protein-sorting mechanisms. Using immunofluorescence microscopy and Western blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA association to the cytoskeleton in erythroblasts, young reticulocytes, and mature erythrocytes using fluorescence imaged microdeformation (FIMD) and observed that GPA underwent dramatic reorganization during terminal differentiation. We discovered that GPA was more connected to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and young reticulocytes than in mature cells. We conclude that skeletal protein association can regulate protein sorting during enucleation. Further, we suggest that the enhanced rigidity of reticulocyte membranes observed in earlier investigations results, at least in part, from increased connectivity of GPA with the spectrin-based skeleton.

Actins↗

PubMedDate not supplied

Effects of base-metal casting alloys on cytoskeletal filaments in cultured human fibroblasts.

PURPOSE: The present study was designed to determine the cytotoxic effects of some widely used dental base-metal casting alloys (Ni-Cr and Co-Cr) on the cytoskeleton in cultured human fibroblasts, and to evaluate whether any structural alteration is associated with the application of these alloys. MATERIALS AND METHODS: Ten specimens from six different alloys were prepared as 5-mm disks. Five of ten samples from each group were polished; the remaining five samples were left sandblasted with 50-microm Al2O3. All samples were directly exposed to human fibroblasts in a 24-well cell culture dish for 120 hours. Then, cells were fixed and stained with antibodies against major cytoskeletal elements--actin, vimentin, and microtubules--by immunofluorescent staining methods. Cells were analyzed in 3-D to document the cytoskeletal alterations using a laser confocal microscope. RESULTS: Disintegration of actin filaments was observed in lamellipodia of fibroblasts by the effect of both polished and sandblasted Ni-Cr and Co-Cr samples, with the exception of the polished Co-Cr alloy (Wirocast). Moreover, intracytoplasmic actin-decorated stress fibers were found bent and occasionally tangled in the sandblasted Ni-Cr (Wiron 99) and Co-Cr alloys (Wirocast and Co-Cr Degussa). Vimentin, a mesenchymal cell intermediate filament protein normally showing an intracellular meshwork pattern, was not affected by any of the polished or sandblasted alloys. Microtubules mainly remained intact in all dental alloy-treated groups. CONCLUSION: Taken together, it is possible to postulate that Ni-Cr and Co-Cr dental alloys, especially sandblasted forms, may have detrimental effects on the actin-based cytoskeleton, at least tested in vitro.

Actins↗

PubMedDate not supplied

Leukocyte chemotaxis.

Monocyte chemotaxis is a function of mononuclear phagocytic system cells that enables migration toward the focus of infection and inhibition of microorganism growth and proliferation. In this paper we review the chemotactic phenomena following Becker's scheme that distinguishes three phases: recognition, transduction and the effector phase. The recognition phase is comprised of the interaction of a chemotactic agent with a receptor located on the cell surface. We cite the multiple chemoattractant presently known, and the mechanisms that regulate this phase, "up-regulation" and "down-regulation" phenomena, and the presence of receptors in low and high-affinity states. The transduction phase comprises all biochemical events produced after internalization of the attractant-receptor complex, whose purpose is triggering the cell motor apparatus. The key event in beginning movement is the increase of cytosol calcium. Alterations in membrane phosphoinositols constitute the initial transductor mechanism in chemotaxis, inositol-triphosphate and diacylglycerol behaving as second messengers. We review the diverse functions of these phospholipids, as well as we do a graphic representation of these (fig 1). Motor phase comprises the movement of the cell toward the chemotactic gradient through activation of the motor cell apparatus. We describe the intervening elements, actin and its regulatory proteins acumentin and gelsolin, actin-fixing protein, myosin and microtubuli, and the four phases of locomotion mechanism: reversible adherence, pseudopode emission, cell polarity and movement that results from an alternance between gel and sol states.

Calcium↗

PubMed1996

2',3'-Cyclic nucleotide 3'-phosphodiesterase binds to actin-based cytoskeletal elements in an isoprenylation-independent manner.

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope-tagged CNP1 and a non-isoprenylated mutant CNP1. Using nonionic detergent extraction, drug-mediated cytoskeletal disruption, and coimmunoprecipitation with an anti-actin antibody, we show that CNP1 is associated with actin-based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c-H-ras, which is also modified by isoprenylation at its carboxyl-terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membranes and the cytoskeleton.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1982

A phosphorylated basic vaccinia virion polypeptide of molecular weight 11,000 is exposed on the surface of mature particles and interacts with actin-containing cytoskeletal elements.

A phosphorylated vaccinia virus structural polypeptide of an apparent molecular weight of 11,000 (p11K) was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used for antibody induction. After purification by antigen affinity chromatography, the immunoglobulins detected only one target of a rather basic nature in two-dimensional immune blotting procedures of total virion proteins. By use of a combination of biological, biochemical, and microscopic techniques, p11K could be located on the surface of those vaccinia virus particles, with "classical" morphology and a buoyant density of 1.27 g/cm3. Upon immunoprecipitation from radioactively labeled infected cells, p11K appeared to be complexed to two additional virus structural proteins, which could be tentatively identified by their molecular weights as precursors for the two major core constituents. When virus assembly was inhibited by rifampin treatment of infected cells, a great part of p11K, either free or in complexed form, was found associated with actin-containing cytoskeletal elements. The ability of p11K to interact with a not-yet-identified, microfilament-associated cellular protein may be related to previous findings showing that assembled vaccinia particles in situ are found in connection with microfilaments. A possible role for the structures precipitated by p11K-specific antibodies in early stages of particle assembly is discussed.

Animals↗

PubMed2005

Cooperativity of actin and microtubule elements during replication of respiratory syncytial virus.

Many paramyxoviruses appear to require cytoskeletal elements for particular steps in the virus life cycle. Measles virus and Sendai virus exhibit a requirement for microtubules in replication in vitro, whereas parainfluenza virus type 3 and RSV require actin for replication. To further elucidate the role of cytoskeletal function and rearrangement in the viral life cycle of RSV, we investigated the efficiency of virus entry, transcription, replication, and budding in the presence of a variety of pharmacological agents that stabilize or depolymerize actin or microtubules. We found that alteration of microtubule or actin function resulted in blocks at entry, formation of cell-associated virus, virus release, local cell-to-cell spread, and syncytium formation. Actin and microtubules act in cooperation to facilitate replication of RSV, although microtubules play a dominant role in the formation of cell-associated virus while actin plays a more prominent role in virus release.

Actins↗

PubMed1981

Overcontraction and excess actin filaments. Basic elements of hypertrophic cardiomyopathy.

Endomyocardial biopsies were taken from the right ventricular aspect of the interventricular septum in three patients with hypertrophic cardiomyopathy and were subjected to electronmicroscopical examination. Longitudinal sections confirmed already well-established findings. In the transverse sections disarray in the arrangement of the actin filaments and expansion of the myosin lattice, indicating clear overcontraction, were observed. The number of actin filaments varied from seven to 14 per hexagon; a number exceeding 12, however, was found in only one case. From our findings we conclude that overcontraction leads to a progressive deviation of the action filaments during systole caused by double overlap. The majority of these mechanisms results in a "self-impeding contraction" of the fibres. Functionally the excess of actin filaments may provide a balance between the unequal forces of contraction.

Actins↗

PubMed1993

Actin is a major structural and functional element of the egg cortex of giant silkmoths during oogenesis.

The cortex and subcortical regions of the developing follicles and eggs of silkmoths are rich in cytoskeletal elements, particularly actin. In situ analysis using [3H]-polyuridylic acid and biotinylated oligo d(T) reveals a pattern of changes in poly(A)+ RNA distribution during oogenesis. The developing pattern of distribution of actin filaments in the ooplasm closely resembles that of poly(A)+ RNA. RNA polymerase II is also associated with the cortical cytoskeleton. Destruction of the actin filaments in the developing oocytes by cytochalasin D randomizes the distribution of mRNA and causes the displacement of RNA polymerase II from the cortex. Rhodamine-conjugated phalloidin and a monoclonal antibody against cytoskeletal actin were used in combination with laser scanning confocal microscopy to examine the details of actin distribution in the oocytes. RNA polymerase II was located in developing oocytes using both anti-Drosophila RNA polymerase II antibody and fluorescein-conjugated amanitin.

Actins↗