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PubMed1991

Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum.

The cytoskeleton of the amoeboid spermatozoa of Ascaris suum consists of major sperm protein (MSP) filaments arranged into long, branched fiber complexes that span the length of the pseudopod and treadmill rearward continuously due to assembly and disassembly at opposite ends of the complexes (Sepsenwol et al., Journal of Cell Biology 108:55-66, (1989)). Examination by video-enhanced microscopy showed that this cytoskeletal flow is tightly coupled to sperm locomotion. The fiber complexes treadmilled rearward at the same rate (10-50 microns/min) as the cell crawled forward. Only fiber complexes with their plasmalemmal ends within a limited sector along the leading edge of the pseudopod underwent continuous assembly. Thus, the location of this sector, which occupies about 50% of the pseudopod perimeter, determined the direction of sperm locomotion. Treatment of sperm with agents that lower intracellular pH, such as weak acids and protonophores, caused the fiber complexes to disassemble completely in 4-5 sec. Removal of these compounds resulted in reassembly of the cytoskeleton in a pattern that mimicked treadmilling in intact sperm. The fiber complexes were reconstructed by assembly at their plasmalemmal ends so that within 30-60 sec the entire filament system reformed and the cell resumed locomotion. Both cytoskeletal reassembly and treadmilling required exogenous HCO3-. These results suggest that variation in intracellular pH may help regulate cytoskeletal treadmilling and thereby play a significant role in sperm locomotion.

Actin Cytoskeleton↗
PubMed1986

The cytoskeleton and insulin secretion.

One of the central, unresolved problems in our understanding of insulin secretion is the way in which stimulus recognition and its associated metabolic events are translated into the mechanical processes of insulin-storage granule movement and extrusion from the cells by exocytosis. In the present article we have examined the structural organization of the B-cell cytoskeleton in detail and have reviewed how drugs that affect the cytoskeleton alter insulin secretion. Available information about the interactions of tubulin, actin, myosin, and actomyosin with insulin-secretory granules is summarized, and a tentative model is proposed to explain how stimulus-effector system coupling might be achieved.

Actin Cytoskeleton↗
PubMed1988

Adhesion patterns and cytoskeleton of rabbit osteoclasts on bone slices and glass.

The ability of osteoclasts (OC) to migrate and resorb bone is thought to be dependent on cytoskeletal function and adhesion. Therefore, we investigated the cytoskeleton and the adhesion patterns of rabbit OC on glass and on devitalized bone slices, using specific antibodies to cytoskeletal elements and fluorescence and interference reflection microscopy. Microtubules (MT) were similar in OC on both substrata, and appeared in a pattern typical of that described for many cells. Multiple centriolar complexes were observed in most OC, either as one large aggregate in the center of the cell or dispersed singly or in small aggregates close to individual nuclei. Staining of microfilaments (MF) was similar on both substrata and appeared primarily as an F-actin network. MF distribution was different in OC associated with resorption lacunae with intense staining over those regions. In the OC on glass, high F-actin staining was detectable at the periphery in dots and rosette-like structures, which also stained for vinculin. The adhesion patterns indicated that OC on glass do not make large focal contacts, but appear to make a few tiny focal contacts that are not associated with the rosette-like structures. Most of the undersurface of the OC appeared either to be involved in close contacts or to be separated by distances of greater than 100 nm from the substratum. These studies indicate that the MF distribution and the adhesion patterns of rabbit OC are typical of motile cells, that the distribution of the cytoskeleton of rabbit OC on glass and on bone slices is similar, and that MF may be involved in the morphological changes associated with resorption.

Actin Cytoskeleton↗
PubMed1988

Vanadate-treated baby hamster kidney fibroblasts show cytoskeleton and adhesion patterns similar to their Rous sarcoma virus-transformed counterparts.

Rous sarcoma virus-transformed baby hamster kidney fibroblasts (RSV/B4-BHK) adhere to a fibronectin-coated substratum by means of dot-like adhesion sites called podosomes in view of their shape and function as cellular feet (Tarone et al.: Exp Cell Res 159:141, 1985). Podosomes concentrate tyrosine-phosphorylated proteins, including pp60v-src, and appear in many cells transformed by oncogenes coding for tyrosine kinases. In this paper we used orthovanadate, an inhibitor of phosphotyrosine phosphatases, in order to increase the cellular concentration of phosphotyrosine and to study whether this treatment induced the cytoskeleton remodeling leading to the formation of podosomes. Indeed, orthovanadate (10-100 microM) induced in a time- and dose-dependent manner the redistribution of F-actin and the formation of podosomes in BHK cells. Cytoskeleton remodeling occurred along with a marked increase of tyrosine phosphorylated proteins. The vanadate effect on the cytoskeletal phenotype was enhanced by the simultaneous treatment of cells with a phorbol ester. Under the latter conditions almost all BHK cells showed podosomes. The vanadate effect was reversible insofar as podosomes and tyrosine-phosphorylated proteins disappeared. Then, vanadate treatment of normal cells induced the cascade of events leading to the cytoskeletal changes typical of transformation and suggested that the transformed cytoskeletal phenotype may be primarily induced by the tyrosine phosphorylation of unknown target(s) operated by endogenous kinases.

Actin Cytoskeleton↗
PubMed1994

Cytoskeleton of cartilage cells.

The cytoskeleton of chondrocytes consists of microfilaments made of actin, microtubules made of tubulin, and intermediate filaments made of a variety of subunits. Actin filaments are not prominent in vivo but may form in vitro. In culture, changes in filament polymerisation are important in determining cell shape, initiating chondrogenesis, and maintaining the chondrogenic phenotype. Microtubules, besides their role in cell division, organise the distribution of organelles and are involved in secretory transport mechanisms in collagen and proteoglycan synthesis. A variety of intermediate filaments may be present, frequently forming large whorled aggregates. The filaments include vimentin, cytokeratins, and glial fibrillary acidic protein. These may occur at different depths in articular cartilage. Vimentin accumulates during development of some fibrocartilages with increased mechanical loading. Together with other elements of the cytoskeleton, intermediate filaments could form part of a mechanotransduction system by which cells respond to external forces and sense changes in their external environment.

Actin Cytoskeleton↗
PubMed2000

Functional properties of sodium channels do not depend on the cytoskeleton integrity.

Several observations suggest an interaction of the sodium channel alpha-subunit with the cytoskeletal structures. However, there is a wide variability in the results of experiments of heterologous expression in Xenopus oocytes and studies on mammalian cells are sometimes contradictory. In general, there has been no direct demonstration that ad hoc large perturbations of the cytoskeleton modify the intrinsic properties of the sodium channels expressed endogenously or heterologously in plasma membranes. We have studied in CHO cells transfected with the rat muscle sodium channel alpha-subunit the effects of two substances expected to produce drastic perturbations of the cytoskeletal structure: Cytochalasin-D, which depolymerizes microfilaments, and Colchicine, which inhibits the microtubules polymerization. We observed no significant differences in the voltage dependence, kinetic parameters and surface density of the expressed sodium channels after treatment of the cells with these substances. We conclude that the two known main components of the cytoskeleton do not interfere directly with the sodium channel function or with the heterologous expression of channels in the cell membrane.

Actin Cytoskeleton↗
PubMed1997

Reorganization of the cortical actin cytoskeleton during maturation division in the Tubifex egg: possible involvement of protein kinase C.

Tubifex eggs undergo a drastic reorganization of the cortical actin cytoskeleton during metaphase of the second meiosis. At the end of the first meiosis, the egg cortex displays only scattered actin filaments and tiny dots of F-actin; during the following 90 min, cortical F-actin gradually increases in amount, becomes organized into foci that are interlinked by actin bundles, and generates a geodesic dome-like organization. In this study, we have characterized this reorganization of the cortical actin cytoskeleton. In living eggs injected with rhodamine-phalloidin at the beginning of the second meiosis, cortical actin assembly (i.e., formation of actin foci and bundles) proceeds normally, but labeled F-actin is not found to be included significantly in the formed cortical actin network, suggesting that the increase in cortical F-actin is not simply ascribable to the recruitment of preexisting actin filaments. Cortical actin assembly can be induced precociously not only by calcium ionophore A23187 but also by a phorbol ester PMA, an agonist of protein kinase C (PKC). Conversely, the formation of actin foci and bundles is inhibited by PKC antagonists, although cortical F-actin increases to some extent in the presence of these inhibitors. Similar inhibition of the cortical reorganization is elicited in eggs whose intracellular free calcium level ([Ca2+]i) has been clamped low by microinjection of a calcium chelator BAPTA. The treatment of BAPTA-injected eggs with PMA results in the formation of actin foci and bundles. An experiment with eggs injected with fluo-3 shows that [Ca2+]i increases during metaphase of the second meiosis. These results suggest that the reorganization of cortical actin during metaphase of the second meiosis requires activation of PKC, which depends on increases in [Ca2+]i.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Annexin V binds to the actin-based cytoskeleton at the plasma membrane of activated platelets.

Immunocytochemical studies demonstrate that annexin V relocates to the plasma membranes of intact stimulated blood platelets. Anti-annexin V antibodies label the cytoplasmic side of the substrate-adherent plasma membrane of mechanically unroofed, glass-activated platelets and colocalize with actin. In addition, crosslinking experiments using detergent-solubilized membranes of activated platelets have identified an 85-kDa complex containing annexin V. The 85-kDa complex is also recognized by antibodies against actin, suggesting that annexin V interacts with actin. In addition, annexin V was found to associate with filamentous actin in the presence of millimolar Ca(2+). Annexin V was also shown by immunofluorescence microscopy to be associated with platelet cytoskeletons, colocalizing with actin in the presence of micromolar Ca(2+). These findings provide the first evidence for annexin V binding to the plasma membrane and to the actin-based cytoskeleton in activated platelets and indicate that annexin V may function in both cytoskeletal and membrane domains.

Actin Cytoskeleton↗
PubMedDate not supplied

Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells.

We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.

3T3 Cells↗
PubMed1987

The cytoskeleton of isolated murine primitive erythrocytes.

Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitive erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.

Actin Cytoskeleton↗
PubMedDate not supplied

The effects of the dynamic state of the cytoskeleton on neuronal plasticity.

The effects of degrading and stabilizing microtubules and microfilaments on the formation of plastic reactions were studied in isolated nerve cells from the mollusk Lymnaea stagnalis. Degradation of the cytoskeleton affected the performance, retention, and repeated acquisition of plastic reactions. Stabilization of microtubules led to the appearance of a relationship between the dynamics of the development and retention of plastic reactions and the series of stimulation. Stabilization of microfilaments led to transient plastic reaction, along with long-term reactions. These results show that rearrangements of the cytoskeleton have a key role in the processes of neuronal plasticity.

Actin Cytoskeleton↗
PubMed1998

Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes.

PURPOSE: Our purpose was to assess the effect of cryopreservation on cytoskeleton of germinal vesicle (GV) mouse oocytes and determine whether irreversible spindle damage and related digyny associated with cryopreservation of metaphase II (MII) oocytes can be avoided. METHODS: The GV oocytes were cryopreserved using a slow-cooling (0.5 degree C/min) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethylsulfoxide supplemented with 0.2 M sucrose and analyzed before and during fertilization by multiple-label fluorescence and differential interference contrast microscopy techniques. RESULTS: When examined after in vitro maturation, the vast majority (> 95%) of cryopreserved and control oocytes displayed normal microfilament and microtubule organization. With respect to barrel-shaped spindle and normal chromosome alignment, no significant differences were observed between cryopreservation (78 and 86%, respectively) and control (85 and 95%, respectively) groups. In fertilization experiments, spindle rotation, formation of the second polar body, and pronuclear migration were displayed by similar percentages of cryopreserved (96, 94, and 37%, respectively) and control (98, 97, and 45%, respectively) oocytes, indicating normal functionality of the cytoskeleton during this period. However, pronuclear formation was significantly inhibited by cryopreservation (81%) compared with controls (100%). Regarding digyny and polyspermy, no significant increase was observed after cryopreservation (3 and 10%, respectively) compared with controls (3 and 6%, respectively). CONCLUSIONS: Cryopreservation of mouse oocytes at the GV stage is particularly advantageous to circumvent the spindle damage and increased digyny noted after cryopreservation of MII oocytes.

Actin Cytoskeleton↗
PubMed2001

The cytoskeleton facilitates a three-dimensional symplasmic continuum in the long-lived ray and axial parenchyma cells of angiosperm trees.

The microtubule (MT), microfilament (MF) and myosin components of the cytoskeleton were studied in the long-lived ray and axial parenchyma cells of the secondary xylem (wood) and secondary phloem of two angiosperm trees, Aesculus hippocastanum L. (horse-chestnut) and Populus tremula L. x P. tremuloides Michx. (hybrid aspen), using indirect immunofluorescence localisation and transmission electron microscopy. MTs and MFs were bundled and oriented axially (parallel to the cell's long axis) within all parenchyma cell types after they had fully differentiated. Additionally, actin and myosin were immunolocalised at the thin-walled membranes of the pits, which linked cells in neighbouring files of both ray and axial parenchyma, and at the pits between axial and ray parenchyma cells themselves. Anti-callose antibody immunolocated the plasmodesmata at the pit membranes, and in the same pattern as that of anti-myosin. Ray cells are important symplasmic pathways between the xylem and the phloem throughout the life of trees. We hypothesise that the MT and MF components of the cytoskeleton in the ray and axial parenchyma cells are involved in the transport of materials within those cells, and, in association with the acto-myosin of plasmodesmata at pit fields, are also important in intercellular transport. Thus, the symplasmic coupling between ray cells, between axial parenchyma cells, and between axial parenchyma and ray cells represents an extensive three-dimensional communication pathway permeating the tree from the phloem through the cambium into the wood. We suggest that this cytoskeletal pathway has an important role in delivery of photosynthate, and mobilised reserves, to the actively dividing cambium, and in the movement of materials to sites of reserve deposition, principally within the wood. This pathway could also have an important role in co-ordinating developmental processes throughout the tree.

Actin Cytoskeleton↗
PubMed1988

Organization and functions of cytoskeleton in metazoan ciliated cells.

Ciliated cells are characterized by a highly organized cytoskeleton which is connected with the ciliary apparatus. The organization of microtubules, microfilaments, and cytokeratin filaments is described and the relationships of each network with the ciliary apparatus are emphasized. Possible functions of such a complex cytoskeleton are discussed.

Actin Cytoskeleton↗
PubMed1994

The plant cytoskeleton.

Particles that can nucleate microtubules in vitro have been isolated from higher plant cells. Observations of living cells injected with fluorescent probes have improved our understanding of plant cytoskeleton dynamics. Despite growing recognition of the need for biochemical studies on cytoskeleton-associated proteins, little progress has been made in this field in the past year, although plant lamins have been isolated and partially characterized.

Actin Cytoskeleton↗
PubMed2005

Protein kinase C and the regulation of the actin cytoskeleton.

Protein kinase C (PKC) isoforms are central components in intracellular networks that regulate a vast number of cellular processes. It has long been known that in most cell types, one or more PKC isoforms influences the morphology of the F-actin cytoskeleton and thereby regulates processes that are affected by remodelling of the microfilaments. These include cellular migration and neurite outgrowth. This review focuses on the role of classical and novel PKC isoforms in migration and neurite outgrowth, and highlights some regulatory steps that may be of importance in the regulation by PKC of migration and neurite outgrowth. Many studies indicate that integrins are crucial mediators both upstream and downstream of PKC in inducing morphological changes. Furthermore, a number of PKC substrates, directly associated with the microfilaments, such as MARCKS, GAP43, adducin, fascin, ERM proteins and others have been identified. Their potential role in PKC effects on the cytoskeleton is discussed.

Actin Cytoskeleton↗
PubMed2006

The state of the actin cytoskeleton determines its association with gephyrin: role of ena/VASP family members.

The role the cytoskeleton plays in generating and/or maintaining gephyrin-dependent receptor clusters at inhibitory synapses is poorly understood. Here, the effects of actin cytoskeleton disruption were investigated in eGFP-gephyrin-transfected cells and hippocampal neurons. While gephyrin was not associated with microfilaments in transfected cells, it colocalized with G-actin and cytochalasin-D-induced F-actin patches. The linker region between the MoeA and MogA homology domains of gephyrin was required for colocalization with F-actin patches and for the binding of gephyrin to ena/VASP, an actin anti-capping factor that, in vitro, caused gephyrin binding to polymerized actin. In hippocampal neurons, treatment with cytochalasin D resulted in the redistribution of the neuronal ena/VASP homologue Mena into actin patches and, at early stages of development, a reduction in the number of gephyrin clusters. Our data suggest that Mena binding to F-actin allows for gephyrin recruitment to the leading edge of uncapped actin filaments.

Actin Cytoskeleton↗
PubMed2004

Genetic modification of the heart: chaperones and the cytoskeleton.

In the past decade, genetic modification has been extensively employed to define (patho)physiological roles of chaperones and the cytoskeleton in the heart, promoting dramatic advances in this field. Both loss-of-function and gain-of-function approaches have been used productively. alphaB-Crystallin (CryAB) is the most abundant small heat shock protein (HSP) in the heart. A missense mutation (R120G) in CryAB that is linked to human desmin-related myopathy (DRM), has proved in transgenic (TG) mice to be causative, likely through compromising the function of both CryAB and desmin filaments and inducing aberrant protein aggregation. For the molecular chaperones, the consensus gained is that up-regulation of each of the HSPs in the heart is protective against insults such as ischemia/reperfusion (I/R) injury. CryAB modulates protein aggregation of abnormal desmin. With respect to the cytoskeleton, disruption of the non-sarcomeric actin linkage at the intercalated discs via overexpressing the VASP-EHV1 domain is sufficient to cause dilated cardiomyopathy (DCM). Up-regulation of microtubule-associated protein 4 (MAP4) results in microtubule densification. Myocyte contractile malfunction characteristic of pressure overload hypertrophy is recapitulated by cardiac-restricted overexpression of MAP4. In contrast, overexpression of desmin filaments by itself is not detrimental to the heart. Although loss-of-function studies have been more limited, ablation of the desmin gene causes mitochondrial dysfunction and apoptosis, resulting in cardiomyopathy in mice. From function studies, abnormal desmin aggregation and disruption of the desmin networks resulting from expression of either mutant desmin or mutant CryAB have been shown to remodel the heart and compromise cardiac function, suggesting their synergistic roles in disease pathogenesis.

Actin Cytoskeleton↗