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To date, the molecular mechanisms underlying the osmoregulation of pollen grains (PGs) related to the maintenance of their water status and allowing pollen tubes (PTs) to regulate concentrations in them of osmolytes and transmembrane water transport remain to be not so far characterized. In the present work, the data on the participation of IAA and ABA in the osmoregulation of germinating in vitro petunia male gametophyte were obtained. It has been established that the growth-stimulating effect of these phytohormones is due to their action on intracellular pH (pHc), the membrane potential of plasmalemma (PM), the activity of PM H(+)-ATPase, K(+)-channels in the same membrane and organization of actin cytoskeleton (AC). Two possible targets of the action of these compounds are revealed. These are represented by (1) PM H(+)-ATPase, electrogenic proton pump responsible for polarization of this membrane, and (2) Ca(2+)-dependent K+-channels. The findings of the present work suggest that the hormone-induced pHc shift is involved in cascade of the events including the functioning of pH-dependent K+-channels. It was shown that the hormoneinduced hyperpolarization of the PM is a result of stimulation of electrogenic activity of PM H(+)-ATPase and the hormonal effects are mediated by transient elevation in the level of free Ca(2+) in the cytosol and generation of reactive oxygen species (ROS). The results on the role of K(+) ions in the control of water-driving forces for transmembrane water transport allowed us to formulate the hypothesis that IAA and ABA stimulate germination of PGs and growth of PTs by activating K(+)-channels. In addition, the studies performed showed that the AC of male gametophyte is sensitive to the action of exogenous phytohormones, with to more extent to the action of IAA. As judged by the action of latrunculin B (LB) the AC may serve as the determinant of the level of endogenous phytohormones that most likely participate in the regulation of the polar growth of PTs impacting on the pool of F-actin in their apical and subapical zones.

On MDCK1 cell monolayer, dynamics of the spatial organization of actin cytoskeleton and dynamics of trans-epithelial electrical resistance (TEER) have been studied upon exposure of arginine-vasopressin (AVP) and protein-kinase A (PKA) activator forskolin. It has been found that exposure to these physiologically active compounds causes fibrillary actin depolymerization (both in apical and in basal cytoplasm) and, simultaneously, significant decrease in the cell monolayer trans-epithelial electrical resistance. TEER decrease indicates the stimulation of ions and water flow across the cell monolayer. In order to clarify pathways of movement of ions and water across MDCK monolayer, we have carried out an immunofluoresence study of claudin 1 and 2 localization in the tight junctions of MDCK ATCC cells (low TEER) and MDCK1 cells (high TEER). We have demonstrated that in the tight junctions of MDCK ATCC cells both claudin 1 and claudin 2 are present. In MDCK1 cells tight junctions, claudin 1 is localized and pore-forming claudin 2 is completely lacking. Under forskolin exposure to MDCK1 cells, no alterations in studied claudins distribution has been found. These data indicate that forskolin-induced TEER decrease is linked with alterations in trans-cellular, not in para-cellular, permeability of monolayer.

Endothelial cells are tightly fitted to each other and lining the interior surface of all vessels of living organism to provide vascular permeability regulation and interchange between the blood circulating in vessels and tissue fluids of those organs in which these vessels are located. In vitro endothelial monolayer conserve it's basic barrier function which is native for vessels endothelium. Based on this fact we used endothelial cells growing in vitro as a model system in experimental studies of cytoskeletal and adhesion cell components interaction. In current paper, cultured human vein endothelial cells monolayer was used to quantify cytoskeleton alterations in the of endothelial cells from spreading and formation of the first cell-cell contacts to confluent monolayer formation. The system of actin filaments formed two different cytoskeletal structures in the cells of venous endothelium: 1) cortical actin network; 2) actin stress fibers (bundles) arranged parallel to the substrate. Two actin isoforms, β- and γ-cytoplasmic (non-muscle) actins, are expressed in endothelial cells. The bundles of actin stress fibers were detected by immunofluorescent staining with antibody against β-actin, whereas antibodies against γ-actin identified cortical and lamellar networks. For assessment of the actin cytoskeleton organization it's fluorescence intensity on the area of 10 μM2 located (1) near the free edge, and (2) in the zone of cell-cell contacts were analyzed. Fluorescence intensity of β-actin structures was higher in the areas of cell-cell contact. The fluorescence of γ-actin structures was more intensive at the leading edges of the lamellae, and was the lowest on the stable edges of the cells with formed cell-cell contacts. The endothelial monolayer formation was accompanied by microtubule system alteration: the number of microtubules increased at the cell edge, and besides the microtubules quantity in the area of already formed cell-cell contact was always higher than in free lamella region.

The analysis of actin cytoskeleton reorganization in rat bone marrow multipotent mesenchymal stromal cells after one hour adhesion to a monolayer of endothelial cell line EA.hy 926 allowed us to identify three types of cells interacting with the endothelial cells. Approximately half of multipotent mesenchymal stromal cells retained a rounded shape, most of them contained large round actin aggregates, had irregular borders and contacted with the surface of the endothelial cells by microvilli or protrusions similar to small lamellae. Almost all other cells were surrounded by narrow lamellae along the entire perimeter. In addition, a small amount.of elongated flattened cells that contacting with endothelial cells by means of focal contacts was observed. Microenvironmental factors such as proinflammatory cytokine tumor necrosis factor α or plasma proteins affected the ratio of stromal cell types, with different types of organization of the actin cytoskeleton in multipotent mesenchymal stromal cells population.

An expression ofintegrins on multipotent mesenchymal stromal cells (MMSCs), isolated from human subcutaneous adipose tissue during long cultivation was studied. Results of the comparative analysis of MMSCs on the 2nd and 17th passages of cultivation revealed considerable distinctions in expression of α1, α4 and α6 integrins on these cells. Strong decrease for 87.2 and 11.2%, quantities of the cells which have been positively stained by AT against CD49a (α1 integrin) and CD49d (α4 integrin), respectively was observed. The share of the cells which have been positively stained by AT against CD49f (α6 integrin) and CD49b (α2 integrin) on the contrary was increased during long cultivation by 9.9 and 2.3%, respectively. The high expression of β1 integrin (98%) on MMSCs, wasn't changed significantly in the course of long cultivation. As a result of induction of MMSCs to a differentiation in the osteogenic direction it was revealed that cells on the 17th passage of cultivation concede by efficiency of formation of cells of bone tissue and extracellular matrix in vitro to cells which induced on the 2nd passage. Thus, long cultivation of MMSCs which is required for building of cellular population after isolation, influences on a cytoskeleton and adhesive abilities of cells that it is necessary to consider when using them for cellular technologies, including for modeling of this or that tissue in three-dimensional scaffolds.

The endothelium lining the inner surface of blood vessels regulates vascular permeability, permitting the exchange between the blood circulating in vessels and tissue fluid, and performs thereby the barrier function. Endothelial cells cultured in vitro retain the ability to perform a barrier function that is inherent in vascular endothelial cells in vivo. Endothelial monolayer in vitro is a unique model system that allows studying the interaction of cytoskeletal and adhesive structures of endothelial cells from the earliest stages of its formation. In this paper we describe and characterize quantitatively the changes in the cytoskeleton of endothelial cells from the time of endothelial cells spreading on the glass and formation of the first contacts between neighboring cells un- til the formation of a functional confluent monolayer. The main type of intermediate filaments of endothelial cells were vimentin filaments. The location of vimentin filaments and their number did not change at different stages of the endothelial monolayer formation, they occupied more than 80% of the cells. The system of actin filaments in endothelial cells was represented by the cortical actin at the cell periphery and bundles of actin stress fibers arranged in parallel. Upon the formation of contacts with neighboring cells the number and thickness of actin filaments increased. In addition, the formation of the endothelial monolayer led to changes in microtubule network, which was evident from the increase in the number of microtubules at the cell edge. Further, at all stages of assembling the endothelial monolayer, the number of microtubules formed at the cell margin in the area of cell-cell contacts exceeded the number of microtubules in the area of the free lamellae.

On the basis of inhibition analysis using inhibitors of protein kinase A and polymerization of microfilament under the influence oftheophylline, GDP and GTP have studied the release of Ca2+ from intracellular stores (ryanodin- and inositol-1 ,4,5-trisphosphate) of native and devitrified porcine oocytes. It is found that theophylline, as well as GDP stimulate the release of Ca2+ from intracellular stores in the native and devitrified oocytes, but a further release of Ca2+ from intracellular stores under the joint action of theophylline and GDP was observed only in native oocytes. Inhibitors of protein kinase A and polymerization of microfilaments in native oocytes have a negative impact on the further release of Ca2+ from intracellular stores under the joint action of theophylline and GDP. In possible transition of Ca2+ between intracellular depots, that stimulated by GDF, but GTF doesn't take part. The received results indicate that disruption of the functioning of the system of microfilaments in oocytes determined by vitrification that assumes a priori difference in the signal transduction pathways in native and devitrified oocytes at diplotene stage.

Gravitropic response is a plant growth response against changing its position relative to the gravity vector. In the present work we studied actin cytoskeleton rearrangements during Arabidopsis root gravitropic response. Two alternative approaches were used to visualize actin microfilaments: histochemical staining of fixed roots with rhodamine-phalloidin and live imaging of microfilaments in GFP-fABD2 transgenic plants. The curvature of actin microfilaments was shown to be increased within 30-60 min of gravistimulation, the fraction of auxially oriented microfilaments decreased with a concomitant increase in the fraction of oblique and transversally oriented microfilaments. Methodological issues of actin cytoskeleton visualization in the study of Arabidopsis root gravitropic response, as well as the role of microfilaments at the stages of gravity perception, signal transduction and gravitropic bending formation are discussed. It is concluded that the actin cytoskeleton rearrangements observed are associated with the regulation of basic mechanisms of cell extension growth by which the gravitropic bending is formed.

Pathogenesis of most myopathies including inherited hypertrophic (HCM) and dilated (DCM) cardiomyopathies is based on modification of structural state of contractile proteins induced by point mutations, such as mutations in alpha-tropomyosin (TM). To understand the mechanism of abnormal function of contractile system of muscle fiber due to Glu180Gly, Asp175 or Glu40Lys, Glu54Lys mutations in alpha-TM associated with HCM or DCM, we specifically labeled alpha-TM by fluorescence probe 5-IAF after Cys-190 and examined the position and mobility of the IAF-TM in the ATP hydrolysis cycle using polarized fluorescence technique. Analysis of the data suggested that the point mutations in alpha-TM associated with hypertrophic or dilated cardiomyopathy caused abnormal changes in the affinity ofTM to actin and in the position of this protein on the thin filaments in the ATPase cycle. Mutations in alpha-TM associated with HCM caused a shift of TM strands to the center of the thin filament and increased a range of tropomyosin motion and affinity of this protein to actin in the ATPase cycle. In contrast, mutations in alpha-TM associated with DCM shifted the protein to the periphery of the thin filament, reduced the amplitude of the TM movement and its affinity for actin. It is proposed that anomalous behavior of TM on the thin filaments in ATPase cycle may provoke the dysfunction of the cardiac muscle in patients with HCM and DCM.

Bacterial are able to invade eukaryotic cells manipulating their own uptake by the host cells phagocytosis, Invasive bacteria can induce this process using extracellular toxins, cell surface ligands and virulence factors injected into the host cell. Two main mechanisms of invasion are recognized: the "zipper" mechanism in which bacteria bind host cell receptors to initiate cytoskeletal rearrangements and membrane extensions necessary for invasion, and the trigger mechanism in which bacteria regulate their own phagocytosis injecting regulatory proteins into the host cell cytoplasm. Most often, the targets in the host cell are signaling pathways not specific for phagocytosis and directly cytoskeleton. In addition, phospholipid composition of the target cell membrane plays an important role in the regulation of bacterial invasion. Thus, the efficiency of invasion is determined not only by bacterial virulence factors, but also by the life cycle, transformation, composition of the membrane, and other physiological characteristics of the host cell. This review describes the various mechanism of bacterial invasion into eukaryotic cells and the factors that may determine the sensitivity of eukaryotic cells to the invasion.

Influenza remains a significant social threat especially regarding the emergence of new mutant or reassortant strains. Measures of prophylaxis do not provide complete and stable protection from infection and the use of antivirals results in high-level occurrence of resistant forms of viruses. Nowadays more and more attention is paid to find new targets for antiviral therapy that are not directly connected with virus proteins but can act indirectly through cellular mechanisms involved in viral replication. This approach requires complete understanding of various cellular pathways used by influenza virus. Here we present a brief overview of interactions between influenza A virus and the cell cytoskeleton. This interaction is initiated from the very beginning of influenza infection--adsorption--and continues with endocytosis, release of viral RNP and its entry into the nucleus. The role of cytoskeleton during the late stages of infection is also of great importance. It takes part in NP translocation from the nucleus to the cytoplasm, virus assembly and budding. The presence of cellular actin in certain influenza virions is therefore not accidental but reflects the peculiarities of interaction between a virus and a host cell.

The presence of actin-binding protein, tropomyosin, shaped as particles or protein complexes that have no bonds with actin structures were found while the analisys of structural rearrangements of actin cytoskeleton. However, their functioning is still unknown. To study the composition and properties of these protein complexes a novel method of their separation from the cells without destroying the structures of the cytoskeleton have been developed. The protein composition of isolated tropomyosin particles has been analised by gel filtration, electrophoresis and Western blotting. They appeared to be a multimolecular complexes of about 700 kDa. Beside the tropomyosin and actin these complexes also contain the Hsp70, Hsp90 and myosin-9 identified by mass spectrometry analisys. Also, under inhibition of deacetylases by trichostatin A, changes in the number of particles and redistribution of tropomyosin between cytosol and cytoskeleton take place along with actin cytoskeleton rearrangements. The results obtained give a reason to assume that these multimolecular complexes may participate in the process of reorganization of the actin microfilaments.

Cholesterol is one of the major lipid components of plasma membrane and it plays an important role in various signaling processes in mammalian cells. Our study focused on the role of membrane cholesterol in organization and dynamics of actin cytoskeleton. Experiments were performed on cultured transformed cells characterized by weakly developed actin network and reduced stress fibers--human embryonic kidney HEK293 cells, epidermoid larynx carcinoma HEp2 cells and mouse fibroblasts 3T3-SV40. Using F-actin labeling with rhodamine-phalloidin, actin cytoskeleton rearrangements were analyzed after sequestration of membrane cholesterol by cyclic oligosaccharide methyl-beta-cyclodextrin, and polyene macrolide antibiotic filipin. In cells treated with methyl-beta-cyclodextrin or filipin, similar processes of actin cytoskeleton reorganization involving filament assembly were revealed. In carcinoma HEp2 cells and fibroblasts 3T3-SV40, cholesterol-sequestering reagents induced intensive stress fiber formation and enhanced cell spreading which corresponded to reversion of transformed phenotype. The rearrangements of cytoskeleton are likely initiated by disruption of lipid raft integrity that is critically dependent on the level of the membrane cholesterol.

We compared spreading of Vero fibroblasts when microtubules were depolymerized or stabilized. After initial attachment cells start blebbing that continues for different time and abruptly transfers into spreading. After spreading initiation, most cells spread in an anisotropic manner through stochastic formation of lamellipodia. A second mode was rapid, isotropic spreading via formation of circular lamellum that occurs in 15% of cells. The rate of spreading was maximal at the beginning and decreased during the first hour according to logarithmic law. After 60 min many cells formed stable efges and started migrating on the substrate. However, cell area slowly continued to increase. Actin bundles are formed 20 min after cell attachment and they first run along cell boundary. This system disassembles within 20-40 min and is substituted with stress fibers crossing the cell. In the isotropically spread cells no actin bunbles are seen. Microtubules in the spreading cells enter into large blebs and all nascent lamella and later form radial array. When MTs has been depolymerized or stabilized blebbing started before cells attached to the substrate and continue much longer than in control cells. In both cases the initial rate of spreading decrease several fold, and remains constant for many hours. After 24 h the mean area occupied by cells with altered MT system was the same as in control. Alteration of MT system had moderate effect on actin system--formation of actin cables started at the same time as in control (within 20 min upon cell attachment), however, they grew even in cells undergoing prolonged blebbing. Actin cables running along cell margin were similar to tat in control cells, but they did not disappear up to 1 h. When stabilized, microtubules form chaotic array: they do not enter blebs and in spread cells run parallel to the cell margin at a distance of 3-5 microm. We conclude that dynamic microtubules speed up completion of blebbing and promote early stages of fibroblasts spreading.

Alpha-actinin 4 (ACTN4) belongs to actin binding proteins of the spectrin superfamily. Structural organisation of actin fibres and focal contacts is considered to be its primary function in a cell. Besides that, nucleocytoplasmic shuffling of ACTN4 and its involvement in nuclear processes were demonstrated. Lately, additional isoforms of ACTN4 resulted from an alternative splicing has been described in various cell types and malignant tumours. In this study, we present investigation of a novel ACTN4 isoform of 80 kDa. The isoform was found in human epidermoid carcinoma cells A431, and it was not detected in human skin fibroblasts, normal human keratinocytes and transformed human embryonic cells HEK293T. Analysis of ACTN4 mRNA in A431 cells showed the presence of a splice variant that lacked the exons 2-8. The deleted exons code two calponin homology domains responsible for ACTN4 binding to F-actin. Intracellular distribution of the described ACTN4 isoform (ACTN4ISO) overexpressed in HEK293T cells differed from that of the full size protein. In the cytoplasm, ACTN4ISO was allocated diffusively with no colocalisation with actin cytoskeleton structures. Intranuclear distribution of ACTN4ISO also differed from that of the full size ACTN4. Nevertheless, immunochemical analysis demonstrated possibility of ACTN4ISO to form heterodimers with the full size protein. Additional investigations of novel isoform interactions with ACTN4 protein partners might clarify its functional features in A431 cells.

A novel 40 kDa protein was detected in native thin filaments from catch muscles of the mussel Crenomytilus grayanus. The MALDY-TOF analysis of the protein showed a 40% homology with the calponin-like protein from the muscle of Mytilus galloprovincialis (45 kDa), which has a 36% homology with smooth muscle calponin from chicken gizzard (34 kDa). The amount of the calponin-like protein in thin filaments depends on isolation conditions and varies from the complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the contact of the protein in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22 degrees C and optimal values of both pH and ionic strength of the extraction solution, total calponin-like protein coprecipitates with thin filaments. At 2 degrees C it remains in the supernatant. The 40 kDa calponin-like protein from the mussel Crenomytilus grayanus has similar properties with smooth muscle calponin (34 kDa). It is thermostable and inhibits the actin-activated Mg -ATPase activity of actomyosin. In addition, the 40 kDa calponin-like protein isolated without using thermal treatment contains endogenous kinases. It was found that the calponin-like protein can be phosphorylated by endogenous kinases in the Ca -independent manner. These results indicate that the calponin-like protein from the catch muscle of the mussel Crenomytilus grayanus is a new member of the calponin family. The role of proteins from this family both in muscle and ponmuscle cells is still obscure. We suggest that the calponin-like protein is involved in the Ca -independent regulation of smooth muscle contraction.

BHK-21 cells were incubated in a medium containing dopamine (DA) and then their catecholamine content evaluated by using the Falck cytochemical method. The significant intensification of cell fluorescence as compared to that one in control preparations was detected; this effect was proportional to DA concentration and exposure duration and was more pronounced in cells in suspension than in those attached to the substrate. Simultaneous ultrastructural investigation has shown that an increased intensity of the cytoplasm fluorescence correlated with the appearance of the dense network of fibrils that were morphologically identified as F-actin microfilaments. Prior blockade of dopaminergic receptors by haloperidol did not change the following DA effect both on the fluorescence intensity and cell ultrastructure. The data obtained suggest that DA chronically acting on the living cells was able to penetrate into the cytoplasm, causing actin polymerization and incorporating into the newly formed actin cytoskeleton. Structurally, this may be manifested by cytoskeleton and its derivative hypertrophy, that could have a substantial effect on general morphology of the cell.

Polymerization of microfilaments and their subsequent rearrangements under control of actin-myosin interactions are two main processes underlined morphogenetic reactions of cells. We studied their role during spreading of normal and transformed REF52tetRas fibroblasts with adjustable ras oncogene expression. Treatment with inhibitors of cell contractility (Y27532 or blebbistatin) led to disappearance of actin bundles and focal adhesions, but both normal and transformed cells preserved high pseudopodial activity. Spreading was considerably accelerated in normal cells and less accelerated in ras-transformed cells under these conditions. When actin polymerization was suppressed with low concentrations of latrunculin A, stress-fibrills and focal contacts were preserved, but lamellipodial activity was lost in normal cells, so spreading was dramatically inhibited. In the case of transformed fibroblasts, actin bundles and focal adhesions virtually disappeared, but pseudopofial activity was not lost and spreading was suppressed to a lesser extent. Therefore, the most essential process in regulation of cell spreading and polarization is microfilament polymerization at the leading edge. Incidentally, ras-transformed cells are less sensitive to inhibitors affecting cytoskeletal structure than non-transformed ones. Possible mechanisms underlying these diversities are discussed.

Investigations on Arabidopsis thaliana--GFP- MAP4 and A. thaliana--GFP-ABD2 plants treated with tubulin and actin inhibitors has shown that stability of cell growth in the distal elongation zone of plant root is achieved by structural interrelation of microtubules and microfilaments. Use of clinorotation and application of inhibitor analysis allowed us to show that gravity force defines the type of such interrelation.

Influence of modulation of cytoskeleton by colchicine, vinblastine and cytochalasine B on contractile reactions of smooth muscles has been investigated by mechanographical method, by the methods of the double sucrose gup junction. Ratio F/G-actin in smooth muscle cells defined a method of a fluorescent microscopy. Microfilaments in a greater degree than microtubule are involved in regulation of reductions caused by hyperpotassic-induced reductions of membrane of smooth muscle segments of the rat aorta and generation of action potentials and reductions smooth muscle cells from guinea pig urethra. Reductions of vascular segments of aorta in rats caused by a hyperosmotic solution depend on condition of microfilaments and microtubules, whereas reductions in isoosmotic striction cells depend on condition of microfilaments. The last are involved in mechanisms of phenylephrine influence on mechanical strain of vascular segments of the rat aorta. Contrary to that, microtubules are involved in stimulation by phenylephrine electric and contractile activity the smooth muscle cells guinea pig urethra. Oppressiof contractile activity of smooth muscle segments of the rat aorta is cAMP-mediated and depends on condition of microfilaments of cytoskeleton, while action potentials and reductions smooth muscle cells of a ureter depend on condition of microtubules.

In this study we investigated the effect of alpha-lipoic acid (ALA) in concentration range 0.7-5.0 mM on the intracellular level of reduced glutathione, the cell cycle phase distribution, the structure of microfilaments and microtubules of normal (3T3) and transformed (3T3-SV40) fibroblasts. We obtained that ALA increased the glutathione content in transformed cells, but did not change its level in normal cells, induced cell cycle arrest of 3T3 cells (but not 3T3-SV40 cells), and disrupted actin microfilaments in cells of both lines. The effect of ALA was compared with N-acetylcysteine (NAC) action. The whole complex of findings allows us to affirm that each of these antioxidants acts on its own target molecules in normal and transformed cells and activates different signal and metabolic pathways in these cells. But at the same time the intermediate steps of ALA and NAC action can be common (alteration of the intracellular level of glutathione, reorganization of actin cytoskeleton, etc.).

We applied a mathematical model to compare the contributions to the myocardium mechanical activity of two a priori feasible variants of the cooperative effect of myosin cross-bridges on the calcium activation of sarcomere thin filaments. One of these variants implies that cross-bridge cooperative influence on the troponin C affinity to calcium is localized within the same functional cluster A7TmTn where the cross-bridge is attached. The second variant is based on the assumption that cross-bridges may affect the affinity of troponin C to calcium in other functional clusters A7TmTn as well (so that the nearer to the cross-bridge a cluster lies, the stronger the cross-bridge affects the affinity of CaTnC complex in this cluster). Each of these two variants and its contribution to the active mechanical performance of the heart muscle during the contraction-relaxation cycle were alternatively assessed in the model. It was found that only the second variant correctly simulates the mechanical activity of the muscle. Thus, the modeling suggests that just this variant of the cooperativity seems to be more feasible.

Actin-containing filaments have been visualized inside the Xenopus oocyte nuclei due to combination of fluorescence and transmission electron microscopy. It has been shown that these filaments contact with nucleoli, spherical bodies and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing latrunculin causes membrane vesiculation in the cytoplasm, and disruption of the nucleoplasm and nuclear envelope integrity. We suppose that actin-containing filaments belong to crucial cell components which are involved in coordination of nuclear-cytoplasmic interactions as well as distribution and transport of intranuclear components in growing Xenopus oocytes.

Endothelial cell barrier dysfunction is often associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction and finally gap formation. At present time the role of microtubules in endothelial cell barrier regulation is not fully understood, however a number of observations allow to assume that microtubules reaction is the extremely important part in development of endothelial dysfunction. These observations have been forced us to examine the role of microtubule system reorganization in endothelial cell barrier regulation. In quiescent endothelial cells microtubule density is the highest in the centrosome region and insignificant near the cell margin. The analysis of microtubules distribution after specific antibodies staining using the method of measurement of their fluorescence intensity has shown that in control endothelial cells the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of an exponential regression. The hormone agent, thrombin (25 nM), causes rapid increase of endothelial cell barrier permeability accompanied by fast decrease in quantity of peripheral microtubules and reorganization of microtubule system in internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress already through 10 min after the beginning of the treatment). Both effects are reversible -- through 60 min after the beginning of the treatment the microtubule network does not differ from normal one, so the microtubule system is capable to adapt for influence of a natural regulator thrombin. The microtubules reaction develops more quickly, than reorganization of the actin filaments system, which responsible for the subsequent changes in the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in a circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.

Transgenic 3T3 fibroblasts have been generated that express either the wild-type KRP or its truncated mutant lacking the C-terminal domain, which primarily contributes to myosin binding of KRP. It was found that KRP-expressing cells display a significantly increased content of myosin filaments and a reduced level of rMLC phosphorylation, whereas the mock transfected cells or cells expressing the C-terminally truncated KRP do not. Our results suggest that (1) KRP promotes the polymerization of myosin II and reduces the rMLC phosphorylation level in cells, (2) KRP acts through direct binding to myosin II, and (3) transgenic 3T3 fibroblasts stably expressing KRP represent a useful and versatile model to study the role of myosin II filament dynamics in cell motility.

Structure and dynamics of actin cytoskeleton play a role ih regulation of cell adhesion, spreading and migration. TRIP6 is a LIM domain-containing protein interacting with many actin-associated proteins and in addition modulating activity of certain transcription factors. To study functions of TRIP6 we inhibited its expression in A549 and A431 cells by short interfering RNAs (siRNAs). The TRIP6 knock-down lead to the increased number and length of stress fibers and to the induction of locomotive phenotype. There was observed decreased number and reorganization of focal adhesions revealed by staining for paxillin, and loss of cell to cell adhesions revealed by staining for E-cadherin. The above changes in cell morphology were accompanied by 2-fold increase in the cell motility rate assessed by the wound healing assay. Thus, down-regulation of TRIP6 in the cell lines used results in increase in the features characteristic to malignant transformation of epithelial cells. Possible mechanisms for the observed effects are discussed.

Published and original data on the organization and ultrastructure of the subsurface (cutaneous) muscle, basal plate, and intercellular matrix of acanthocephalans (spiny-headed worms) were analyzed and reviewed. Similar to flatworms, the basal plate and the intercellular matrix of acanthocephalans proved to be derived from the cutaneous muscle. Specific structure of acanthocephalan cutaneous muscle (the lacunar system, "nuclear secretion", specific block arrangement of myofilaments, etc.) allows us to consider it as a special kind of smooth muscle tissue.

The effect of N-ethylmaleimide on the polymerization of myorod, a protein of molluscan smooth muscles, which is colocalized with myosin on the surface of paramyosin core of thick filaments and is a product of the alternative splicing of the gene of heavy myosin chains, was studied. It was shown that myorod modified by N-ethylmaleimide completely loses the polymerization ability but acquires the ability to aggregate in the presence of Mg2+. At the same time, treatment of molluscan myosin with N-ethylmaleimide did not affect its polymerization. It was supposed that the effect of N-ethylmaleimide on myorod polymerization is related to the modification of the myorod SH domain containing Cys722.

Architectonics of the fibrillar actin in cultures of human endothelium cells (EC) during and after extended (144 hrs.) clinostatting was examined under the fluorescent microscope. In control EC cultures, F-actin was composed of a network of filaments located both in the center and along the periphery of cells. One hour of rotation did not visibly affect the actin skeleton, however, in two hours there were cells with filaments displaced toward the membrane. Twenty-four hours of rotation caused total remodeling of the cytoskeleton so that practically the whole population of thin filaments had migrated toward the periphery. In most of the cells the intercell contact area formed undulating edges rich in F-actin. A similar picture could be seen after 144 hrs. of rotation. The actin skeleton partially recovered in 4 hrs. and regained the normal structure in 24 hrs. of rehabilitation. Same period was required for cells to restore their structure completely following the extended clinostatting. These data suggest sensitivity of the EC cytoskeleton to changes in the gravity vector. Remodeling of the actin skeleton in a changed gravity is reversible and proceeds more rapidly than recovery.

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.

Effects of long-term simulation of hypogravity on actin cytoskeleton and cell migration were investigated in cultured human endothelium cells (EC). In control, F-actin resided predominantly on the periphery of cell forming an array of parallel bundles with "dense bodies" along the edge. A small number of actin cable fibers was found in the center. Already after 1-2 hrs of clinostatting at 5 RPM the cell cytoskeleton showed actin filament thinning and displacement toward the cell edges. In subsequent 6-18 hrs, almost all actin fibers had left the center part of EC and had ranged themselves in a continuous F-actin line in the intercellular contact area. In most cases, these changes resulted in the so-called "ruff-edge". Since both the disappearance of cable fibers and formation of the "ruff-edge" add to the cell migration activity, this parameter was studied with the would-healing model. According to our data, 24-48 hrs of exposure to hypogravity stimulates cell migration and expedites 2-3 times reparation of mechanically damaged monolayer. The results suggest that effects of hypogravity on cultured human EC are likely to be consequent to alterations in the activity of protein kinase C and/or adenylate cyclase involving many members of the cellular metabolism.

Interrelations among changes in 11-oxycorticosteroids (11-OSC) in blood plasma, catecholamines (CA), and acetylcholinesterase (AChE) activity in nerve fibres of the thymus, as well as qualitative content and proliferative capacity of thymic and blood cells under the effect of the rat recombinant interleukin-1 beta (IL-1 beta) normally and under conditions of hypothalamic-pituitary-adrenocortical depression, were studied. The changes were the most obvious within 15 and 120 min following the IL-1 beta administration: elevation of the 11-OSC, increase in the CA content, and decrease in the AChE activity in thymic nerve fibres. Preliminary administration of dexamethasone prevented the effects.

Abrikosov's tumour was studied light- and electron-microscopically and immunohistochemically with the use of antibodies against desmin, myoglobin, vimentin, cytokeratin, glial fibrillar acid protein, neuron-specific enolase, S-100 protein, neurofilaments (68 kd), lysozyme, alpha-1-antichymotrypsin, alpha-1-antitrypsin, structural components of extracellular matrix (collagen I, II, III, IV, V types and fibronectin). The presence of neurofilaments, apart from vimentin, S-100 protein and small amount of neurospecific enolase, in the tumour cells is demonstrated for the first time. Neurofilament expression by tumour cells and the lack of the basal membrane collagen (type IV) in the extracellular matrix indicate the cell differentiation different from that of Schwann cells. A similar cell immunophenotype is typical for tumours of sympathetic ganglion and paraganglion structures. Both these results and literature data prove phenotypic heterogeneity of the granular cell tumours and their histogenetic link with the cells of nervous comb.

Microfilaments were shown to be involved in maintaining spatial organization of the Xenopus laevis oocyte by using injections of DNAse I, which selectively destroys F-actin. An original method has been proposed for visualization of polymerized actin at the electron microscope level. Standard histological sections (5-6 um) were deparaffinized, treated with gold-labelled phalloidin, dehydrated, and embedded in epoxy resins using gelatin capsules placed onto sections. After resin polymerization, the section separated from the slide in liquid nitrogen was cut in ultrathin sections on an ultratome. This method allows to by-pass the cutting on an ultracryotome and to study the distribution of polymerized actin in any region of the oocyte at the electron microscope level. The method allows to make the light immunohistochemistry data more precise. We have shown using this method the presence of F-actin not only in the cortex, as was established earlier, but also in deep layers of the ooplasm. Some aspects of the intracellular matrix organization are discussed.

Spread fibroblasts contain a dense microfilament sheath under the dorsal cell surface in the endoplasmic region. The formation of the sheath during spreading of mouse embryo fibroblasts was studied using electron microscopy of platinum replicas. At the first stages of spreading the actin meshwork comprising the pseudopodial cytoskeleton arises at the cell edges. The actin of unattached pseudopodia moves centripetally and forms a circular microfilament bundle at the endoplasm periphery. Simultaneously, the microfilament cortex in the endoplasm appears to disassemble. Due to a continuous supply of polymerized actin from the periphery to the circular bundle the latter becomes wider to cover gradually the endoplasm and to form the microfilament sheath. Anchoring of centripetally moving microfilaments at the sites of cellular contacts with the substratum leads to the formation of radial actin bundles.

To release Z-discs of skeletal muscles myofibrils from actin microfilaments, I--Z--I-brushes (complexes of Z-discs and thin filaments) were treated with DNAse I-both in suspension and on electron microscopical grids. It was shown that such a treatment resulted in depolymerization of actin filaments. The preparations obtained were heterogeneous and contained I--Z--I-brushes with shorter actin filaments and single Z-discs. The structure of Z-discs released from actin filament remained intact. Therefore these preparations may be used in studies on regulation of actin microfilaments assembly.

By the use of electron microscopy and optical diffraction the interaction of F-protein (phosphofructokinase) with F-actin and reconstructed thin filaments has been revealed. F-protein distributes itself along the filament bundles at regular intervals of 36-38 nm.