Reorganization of actin cytoskeleton in 3T3-SV40 cells and their sensitivity to lysis by natural killer cells

The present work was aimed to examine whether the actin reorganization of 3T3-SV40 cells influences their sensitivity to natural killer (NK) cells activity. The effects of N-acetylcystein (NAC) and latrunculin B, actin depolimerizator, on both cellular parameters were studied. Experiments with NAC demonstrated that 3T3-SV40 sensitivity to NK cells activity remained unchanged under the disordered microfilaments but decreased upon the appearance of structured stress-fibres. The data on latrunculin B action resulted in the opposite conclusion: the more microfilaments disorganization in the presence of latrunculin B the lesser 3T3-SV40 sensitivity to lysis by NK cells. These facts suggest that relations between microfilament integrity in 3T3-SV40 cells and their sensitivity to NK cells are rather independent. The latter confirms our previous conclusion (Gamaley et al., 2006). Decrease in 3T3-SV40 sensitivity to NK cells activity accompanied by actin reorganization resulted from both latrunculin B and NAC action suggests changes in cellular surface, which ultimately lead to inactivation (or loss) of the molecules being activating signals to NK cells.     

Reorganization of actin cytoskeleton in 3T3-SV40 cells and their sensitivity to lytic activity of natural killer cells

The goal of the present work was to find out whether the actin reorganization of 3T3-SV40 cells affects their sensitivity to the activity of natural killer (NK) cells. The effects of N-acetylcysteine (NAC) and the inhibitor of actin depolymerization, latrunculin B on both cellular parameters were studied. Experiments with NAC demonstrated that the sensitivity of 3T3-SV40 cells to the NK cell activity remained unchanged with disordered microfilaments, but decreased upon the appearance of structured stress-fibers. With respect to latrunculin B action, the opposite conclusion has been drawn, which is that the more microfilaments disorganization in the presence of latrunculin B, the lower the 3T3-SV40 sensitivity to lysis by NK cells. These facts suggest that relations between microfilament integrity in 3T3-SV40 cells and their sensitivity to NK cells are rather independent, which confirms our previous conclusion (Gamaley et al., 2006). A decrease in the 3T3-SV40 sensitivity to the NK cell activity accompanied by actin reorganization resulting from the influence of both latrunculin B and NAC suggests changes in the cellular surface that ultimately lead to the inactivation (or loss) of molecules that are activating signals to NK cells.     

Effect of alpha-lipoic acid on 3T3 and 3T3-SV40 fibroblasts: Comparison with N-acetylcysteine

In this study, we have investigated the intracellular level of reduced glutathione, cell-cycle phase distribution, and microfilament and microtubule structures in normal (3T3) and transformed (3T3-SV40) fibroblasts exposed to alpha-lipoic acid (ALA) in concentrations of 0.7–5 mM. It was found that ALA treatment increased the glutathione content in transformed cells, but did not affect its level in normal cells; moreover, it also induced the cell-cycle arrest of 3T3 cells (but not 3T3-SV40 cells) and disrupted actin microfilaments in cells of both lines. The ALA effect was compared to that of N-acetylcysteine (NAC), another antioxidant we examined previously. The findings allow us to assert that each of these antioxidants impacts on distinct target molecules in normal and transformed cells and activates different signal and metabolic pathways in these cells. However, intermediate steps of ALA and NAC action may be common (altered intracellular level of glutathione, reorganization of actin cytoskeleton, etc.).     

Coupled Activation of Mechanosensitive and Calcium-Dependent Potassium Channels in 3T3 and 3T3-SV40 Cells

Using the patch–clamp method, mechanosensitive regulation of ion channels was studied in cultivated 3T3 and 3T3-SV40 fibroblasts. The activity of mechanosensitive cation channels with a conductivity 25 pS in response to plasma-membrane stretching was observed in both cell lines. Despite obvious differences in the actin network in normal and transformed cells, the threshold values of the stimulus required for the channel activation were close and were approximately 55 mm Hg. The frequency of channels was significantly higher in transformed 3T3-SV40 fibroblasts than in their untransformed 3T3 analogs. Coupled activation of mechanosensitive calcium-permeable channels and potassium calcium-controlled channels was found in both cell lines. The analysis of flows through single channels allows to detect functional interaction of different channels: stretch-induced local calcium entry activates potassium channels that do not have their own mechanosensitivity. The results of a comparative study show that there is a fundamental similarity between the ion mechanisms of cellular mechanotransduction in normal and transformed fibroblasts. The quantitative differences, first of all, concern the level of functional activity of mechanosensitive channels that provide the development of the local calcium signal in the near-membrane cell region.     

The characteristics of actin cytoskeleton structure and its rearrangements by extracellular matrix proteins in normal, immortalized and transformed rat fibroblasts

Comparative analysis of actin cytoskeleton structure in rat embryonic fibroblasts, E1A-immortalized and E1A + cHa-ras-transformed cells has been carried out. A decrease in adhesiveness and the rate of changes in actin cytoskeleton structures was shown to correlate with the level of morphological transformation of cells. E1A + cHa-ras-transformants show the lowest adhesiveness and complete disorganization of actin structures. Cultivation on serum-free media promoted disassembling of actin cytoskeleton structures in a small part of normal fibroblast population, only in a few immortalized cells, but exerted no influence on transformed cells. The influence of immobilized extracellular matrix proteins fibronectin, laminin and collagens type I and III on actin cytoskeleton structure in normal, immortalized and transformed fibroblasts was studied. Transformed cells spread on fibronectin completely restored highly organized actin structures, displayed a lot of stress fibers and focal contacts. The use of laminin revealed differences in locomotion between normal and transformed cells. Normal, immortalized and transformed fibroblasts spread on fibronectin and laminin demonstrate some peculiarities in actin cytoskeleton structures as a result of specificity of ligand-receptor interaction. Cells spread on fibronectin have polygonal shapes, many stress fibers and focal contacts, whereas cells spread on laminin are highly polarized and develop broad lamellae filled with actin microfilament meshwork. Collagens type I and III can affect adhesive properties and actin cytoskeleton structure in all cell lines studied only slightly, in comparison with fibronectin and laminin.     

The role of nod factor substituents in actin cytoskeleton rearrangements in Phaseolus vulgaris

In order to define the symbiotic role of some of the chemical substituents in the Rhizobium etli Nod factors (NFs), we purified Nod metabolites secreted by the SM25 strain, which carries most of the nodulation genes, and SM17 with an insertion in nodS. These NFs were analyzed for their capabilities to induce root hair curling and cytoskeletal rearrangements. The NFs secreted by strain SM17 lack the carbamoyl and methyl substituents on the nonreducing terminal residue and an acetyl moiety on the fucosyl residue on the reducing-terminal residue as determined by mass spectrometry. We have reported previously that the root hair cell actin cytoskeleton from bean responds with a rapid fragmentation of the actin bundles within 5 min of NF exposure, and also is accompanied by increases in the apical influxes and intracellular calcium levels. In this article, we report that methyl-bearing NFs are more active in inducing root hair curling and actin cytoskeleton rearrangements than nonmethylated NFs. However, the carbamoyl residue on the nonreducing terminal residue and the acetyl group at the fucosyl residue on the reducing terminal residue do not seem to have any effect on root hair curling induction or in actin cytoskeleton rearrangement.     

Pasteurella multocida toxin stimulates mitogenesis and cytoskeleton reorganization in Swiss 3T3 fibroblasts

Pasteurella multocida toxin (PMT) causes cytoplasmic retraction in epithelial cells, activates osteoclast neoformation, and is a potent mitogen for Swiss 3T3 fibroblasts. In the present study designed to further investigate the effects of PMT on cell shape and proliferation, we report that the mitogenic effect of affinity-purified PMT on quiescent 3T3 cells was even superior at 5 ng/ml to that of fetal bovine serum or bombesin. This positive effect was inhibited by heat denaturation and methylamine treatment (this agent blocks internalization). Preincubation of PMT with gangliosides GM₁, GM₂, or GM₃ counteracted its effect on DNA synthesis, suggesting that the toxin binds to GM-type ceramides on target cells. The distribution of F-actin was analyzed in control/treated cells using FITC-conjugated phalloidin. In comparison with FBS and bombesin, PMT triggered a more rapid and profound reorganization of cortical actin into prominent stress fibers after only 5–10 min. This event lead to the retraction of cells after only 30 min and ultimately to the induction of mitotic figures. Interestingly, methylamine blocked the effects of PMT on stress fiber formation and cell retraction but not the ruffling response, suggesting that some early events may not require toxin internalization. In summary, these findings indicate that PMT concomitantly exerts a strong mitogenic activity and a rapid stimulation of cytoskeletal rearrangements, possibly after binding to membrane gangliosides and subsequent internalization. We propose that this toxin could be used in the future as a defined inducer of transduction signals involved in cellular proliferation and control of cell shape. © 1996 Wiley-Liss, Inc.     

Benzamide-induced changes in the actin cytoskeleton in human skin fibroblasts.

The incubation of human skin fibroblasts in the presence of 10 mM benzamide in Joklik's modification of Eagle's Minimal Essential Medium caused an extensive reorganization of actin filaments. The disappearance of stress fibers and changes in cell morphology were observed, whereas no changes in the microtubule architecture were noticed. The observed effects appeared fully reversible within 3 hours after the removal of benzamide. The results are discussed in relation to the two known activities of benzamide as an anaesthetic and an inhibitor of ADP-ribosylation.     

N-acetylcysteine reduces transformed 3T3-SV40 fibroblast sensitivity to lysis by natural killer cells

We have shown that 18-20 h cultivation of transformed mouse fibroblasts 3T3-SV40 in the presence of antioxidant, N-acetylcysteine (NAC, 10 mM), did not change their sensitivity to lysis by natural killer (NK) mouse splenocytes. However, in 18-20 h after NAC removal 3T3-SV40 cells demonstrated resistance to NK cell activity. The cytotoxicity index (CI) was reduced up to 4.6 +/- 2.4 % (in comparison with the control value 31.8 +/- 2.4 %) approximating to the value in non-transformed 3T3 mouse fibroblasts. Normal 3T3 cells were resistant to NK action in all experimental conditions (CI varied within 0.7-5.3 %). These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect may be due to the NAC-induced modifications of the cell surface and extracellular matrix proteins.     

Ethanol increases p190RhoGAP activity, leading to actin cytoskeleton rearrangements

We previously reported that cells chronically exposed to ethanol show alterations in actin cytoskeleton organization and dynamics in primary cultures of newborn rat astrocytes, a well-established in vitro model for foetal alcohol spectrum disorders. These alterations were attributed to a decrease in the cellular levels of active RhoA (RhoA-GTP), which in turn was produced by an increase in the total RhoGAP activity. We here provide evidence that p190RhoGAPs are the main factors responsible for such increase. Thus, in astrocytes chronically exposed to ethanol we observe: (i) an increase in p190A- and p190B-associated RhoGAP activity; (ii) a higher binding of p190A and p190B to RhoA-GTP; (iii) a higher p120RasGAP-p190A RhoGAP complex formation; and (iv) the recruitment of both p190RhoGAPs to the plasma membrane. The simultaneous silencing of both p190 isoforms prevents the actin rearrangements and the total RhoGAP activity increase triggered both by ethanol. Therefore, our data directly points p190RhoGAPs as ethanol-exposure molecular targets on glial cells of the CNS.     

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.     

Density inhibition of motility in 3T3 fibroblasts and their SV40 transformants

A dramatic decrease in cellular motility (measured by means of the augmented diffusion constant, D^∗) was observed with increasing cell area densities of 3T3 fibroblasts. This phenomenon was named density inhibition of motility, and a quantitative measure of this effect, the coefficient of density inhibition of motility, was proposed. Control experiments precluded depletion of a nutritional “locomotion factor(s)” as an explanation for the observed decrease in 3T3 motility. By contrast, SV40 transformants exhibited negligible density inhibition of motility. Data and arguments were presented in support of the hypothesis that the strong density inhibition of motility observed for 3T3 reflected a strong mutual adhesivity of these cells, and that the weak density inhibition of motility observed for SV3T3 reflected a correspondingly weak mutual adhesivity.     

Reorganization of the actin cytoskeleton in the protruding lamellae of human fibroblasts.

To investigate the mechanisms of protrusion in vertebrate cells, the primary event in cell motility, human fibroblasts were treated with neomycin, an inhibitor of the phosphatidylinositol cycle, to induce protrusion. Changes in cell motility and the cytoskeleton were examined by video, fluorescence, scanning electron, and confocal microscopy and by cytofluorometry. Protrusion in neomycin-treated human fibroblasts is correlated with a transient overall decrease in F-actin followed by an increase in F-actin at the leading edge of the protruding lamella. In growing lamellae, F-actin is organized in a marginal band at the leading edge. Although actin is present in the lamella behind the leading edge, very little of it is F-actin. Scanning electron microscopy of detergent-extracted cells reveals a band of dense filaments at the leading edge, corresponding to the marginal band of F-actin seen in fluorescently labeled cells, and a sparse population of short, fragmented filaments, in the rest of the lamella. Gelsolin is colocalized with F-actin in the marginal band and is also present in the lamella where F-actin is largely absent. The data support the hypothesis that the protrusion is initiated by the breakdown of cortical actin filaments, possibly mediated by gelsolin, whereas expansion of the protrusion requires de novo polymerization of actin filaments at the leading edge.     

The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts

Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE₂ release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PGE₂ release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PGE₂ release. In fact, PGE₂ release was increased threefold in the polymerization inhibited cells and this PGE₂ release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PGE₂ release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PGE₂ release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton. mechanotransduction; cell mechanics     

Effects of actin cytoskeleton rearrangements on channel activation by calcium entry into A431 cells

Using patch clamp and ion-selective fluorescence dye techniques, we investigated the influence of actin cytoskeleton rearrangements on the activity of calcium entry channels in plasma membrane of human carcinoma A431 cells. It is shown that disruption of actin microfilaments by cytohalasin D has no significant effect on calcium release from the stores and its entry from the extracellular space. It also does not interfere with the activation of inositol 1,4,5-trisphosphate (IP3)-dependent high-selective low-conductance calcium channels Imin. The treatment of cells with calyculin A induces the formation of actin filament layer beneath plasma membrane and also inhibits Imin activation and calcium entry through the plasma membrane, though calcium efflux from the stores was nearly unchanged. Thus, it is concluded that calcium signalling in A431 cells can be modulated by actin cytoskeleton rearrangements, and may be well described in terms of "conformational coupling" model.     

IpaA targets beta1 integrins and rho to promote actin cytoskeleton rearrangements necessary for Shigella entry

Shigella invasion into the colonic epithelium involves many steps including the formation of large membrane protrusions by the epithelial cells that facilitate bacterial engulfment. IpaA, a Shigella protein secreted into target cells upon cell contact induces a loss of actin stress fibers in cells and promotes the reorganization of actin at the site of entry. The mechanism for this is not known but is thought to involve recruitment of the focal adhesion protein vinculin to IpaA. Here we have examined the mechanism for the effects of IpaA on the actin cytoskeleton. We show that IpaA-induced loss of actin stress fibers and cell rounding do not require vinculin expression or an intact vinculin binding site on IpaA. Rather, we find that cells expressing IpaA exhibited elevated Rho activity and increased myosin light chain phosphorylation. In addition, IpaA decreases integrin affinity for extracellular matrix ligands by interfering with talin recruitment to the integrin cytoplasmic tail. The combination of these two effects, namely weakened adhesion and increased contractility, account for the loss of actin stress fibers and cell rounding observed in cells exposed to IpaA.