Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells

Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.     

Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts

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.     

Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.     

Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria

We describe two subpopulations of actin antibodies isolated by affinity chromatography from a polyclonal antibody to chicken gizzard actin. One subpopulation recognizes gamma actins from smooth muscle and nonmuscle cells, but does not recognize alpha actin from skeletal muscle. The other subpopulation recognizes determinants that are common to alpha actin from skeletal muscle and the two gamma actin isotypes. Neither antibody recognizes cytoplasmic beta actin. Both antibodies recognize only actins or molecules with determinants that are also present in actins. By immunofluorescence we found that the anti-gamma actin colocalizes with mitochondria in fibers of mouse diaphragm, and that it does not bind detectably to the 1 bands of sarcomeres. The antibody that recognizes both alpha and gamma actins stains 1 bands intensely, as expected. We interpret these observations as preliminary evidence for selective association of gamma actin with skeletal muscle mitochondria and, more broadly, as evidence for subcellular sorting of isoactins.     

Ultrastructural cytoskeleton alterations and modification of actin expression in the NIH/3T3 cell line after transformation with Ha-ras-activated oncogene.

Cytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Ha-ras-activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine-phalloidin and with a fluorescinated anti-non-muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down-regulation of alpha-actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of alpha-actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between alpha-actin down-regulation and stress fiber loss.     

Behavior of a fluorescent analogue of calmodulin in living 3T3 cells

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.     

12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodamine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. The enhancement of binding of the beads by TPA was suppressed by addition of an adhesion-inhibitory peptide (Gly-Arg-Gly-Asp-Ser-Pro). Treatment with TPA did not enhance nonspecific binding of beads coated with heat-denatured bovine serum albumin. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.     

Change in the topographical distribution of GM3 during cell spreading and growth: immunostaining with monoclonal antibody against GM3

A monoclonal antibody, M2590, that recognizes hematoside (GM3) was used to analyze the immunostaining localization of GM3 of the surface of transformed and non-transformed hamster embryo fibroblasts and B16 melanoma cells. The reactivity of GM3 with the antibody changed markedly depending on the cell density. At the sparse density cells were clearly made visible by the antibody, but at the confluency the accessibility of the antibody to GM3 was greatly decreased. This density dependent change in the reactivity of GM3 was found for both normal and transformed cells. The staining pattern of GM3 was examined in relation to the actin fibers made visible with NBD-Phallacidin during cell spreading. When the cells were still round, the GM3 on microspikes or blebs was highly reactive with the antibody, and by the time cells showed circumferential staining of their actin fibers, GM3 had been distributed over the entire cell surface as punctuated spots. GM3 also was visible in substrate attachment materials (SAM). Trypsin treatment of SAM diminished the reactivity of GM3 with the antibody. The antibody did not inhibit cell attachment or spreading on a substratum coated with fibronectin or laminin.     

The distribution of actin in non-muscle cells : The use of actin antibody in the localization of actin within the microfilament bundles of mouse 3T3 cells

Living mouse 3T3 cells display a complex array of fibrous structures which are visible with phase contrast, Nomarski and polarized light optics. When cells are fixed and stained for indirect immunofluorescence with actin antibody, the same fibers show intense fluorescence indicating that they contain actin. Electron microscopy reveals that these fibrous structures consist of submembranous bundles of microfilaments located primarily on the attached side of the cells. The results are discussed in terms of the intracellular localization of a possible submembranous contractile system involved in motile activities such as cell locomotion.     

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     

Live cell imaging of mitochondrial movement along actin cables in budding yeast

BACKGROUND: Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. RESULTS: We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. CONCLUSIONS: Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.     

A p85 subunit-independent p110alpha PI 3-kinase colocalizes with p70 S6 kinase on actin stress fibers and regulates thrombin-stimulated stress fiber formation in swiss 3T3 cells

The signaling pathways linking receptor activation to actin stress fiber rearrangements during growth factor-induced cell shape change are still to be determined. Recently our laboratory demonstrated the involvement of p70 S6 kinase (p70(s6k)) activation in thrombin-induced stress fiber formation in Swiss 3T3 cells. The present work shows that thrombin-induced p70(s6k) activation is inhibited by the PI 3-kinase inhibitors wortmannin and LY-294002. These inhibitors also significantly reduced thrombin-induced stress fiber formation, demonstrating a role for PI 3-kinase activity in this process, most likely upstream of p70(s6k). Furthermore, the p110alpha form of PI 3-kinase was localized to actin stress fibers, as was previously shown for p70(s6k), as well as to a golgi-like distribution. In contrast, PI 3-kinase p110gamma colocalized with microtubules. The PI 3-kinase p85 subunit, known to be capable of association with p110alpha, was present in a predominantly golgi-like distribution with no presence on actin filaments, suggesting the existence of distinctly localized PI 3-kinase pools. Immunodepletion of p85 from cell lysates resulted in only partial depletion of p110alpha and p110alpha-associated PI 3-kinase activity, confirming the presence of a p85-free p110alpha pool located on the actin stress fibers. Our data, therefore, point to the importance of subcellular localization of PI 3-kinase in signal transduction and to a novel action of p85 subunit-independent PI 3-kinase p110alpha in the stimulation by thrombin of p70(s6k) activation and actin stress fiber formation.     

Localization of ryanodine receptor 3 in the sinus endothelial cells of the rat spleen

The ultrastructural localization of ryanodine receptors (RyR) in sinus endothelial cells of the rat spleen was examined by confocal laser scanning and electron microscopy by using isoform-specific antibodies to each of the RyR isoforms. Immunofluorescence microscopy of tissue cryosections revealed RyR3 to be localized, with a strand-like form, in the superficial layer and within the cytoplasm of endothelial cells. Antibodies to RyR1 and RyR2 did not react indicating RyR3 was the predominant isoform. RyR3 was observed over the cortical layer of actin filaments in the apical part and beneath stress fibers in the basal part of the endothelial cells. The distribution of Ca²⁺-storing tubulovesicular-structures within endothelial cells was established by tissue sections treated with osmium ferricyanide selectively to stain the sarcoplasmic reticulum and transverse tubules in muscle cells; electron microscopy revealed densely stained tubulovesicular structures located throughout the sinus endothelial cells and interconnected at various sites. These structures closely apposed the plasma membrane at the apical, lateral, and basal surfaces of the cells and occasionally ran closely parallel to the plasma membrane and near to the mitochondria. Immunogold electron microscopy revealed RyR in the membranes of the nucleus, tubulovesicular structures, and subplasmalemmal cisternae. In the subplasmalemmal cisternae at the apical, lateral, and basal surfaces, RyR was detected on the membranes near to the plasma membrane. Labeling was also present on the membranes of tubulovesicular structures near to caveolae and on the cristae of the mitochondria. Thus, RyR probably participates in Ca²⁺ signal transduction and/or mechanosignal transduction in sinus endothelial cells.This work was supported by Grant-in-Aid for Scientific Research (C), Japan.     

Effect of sodium butyrate on actin distribution in rat 3Y1 fibroblasts in monolayer culture

We studied the effect of sodium butyrate, a potent G1/G2-arresting agent, on actin distribution in rat 3Y1 fibroblasts in monolayer culture by fluorescence microscopy of cells stained with 7-nitrobenz-2-oxa-1, 3-diazole phallacidine (NBD-Ph). When randomly proliferating cells were arrested mainly in G1 phase with butyrate, a reversible overaccumulation of cellular net protein occurred. In the G1-arrested cells, actin markedly accumulated at the margin of cells, and a network structure of actin stress fibers appeared. When density-arrested cells were replated sparsely and rearrested in the G1, early S, and G2 phases with butyrate or hydroxyurea, the actin network was observed extensively in the cells arrested in the G1 and G2 phases with butyrate. These results agree with our previous results indicating the existence of some physiological similarity between cells in the G1 and G2 phases and suggest that actin distribution somehow depends on the phases of the cell cycle. The actin profiles observed by the NBD-Ph staining were confirmed by transmission electronmicroscopy (TEM) of negatively stained whole cells. TEM further revealed that electron-dense amorphous materials were present at crossing points in the network but rarely present on interconnecting microfilament bundles.     

A retention mechanism for distribution of mitochondria during cell division in budding yeast.

Mitochondria are indispensable for normal eukaryotic cell function. As they cannot be synthesized de novo and are self-replicating, mitochondria must be transferred from mother to daughter cells. Studies in the budding yeast Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately after bud emergence, interact with the actin cytoskeleton for linear, polarized movement of mitochondria from mother to bud, but are equally distributed among mother and daughter cells [1] [2] [3]. It is not clear how the mother cell maintains its own supply of mitochondria. Here, we found that mother cells retain mitochondria by immobilization of some mitochondria in the 'retention zone', the base of the mother cell distal to the bud. Retention requires the actin cytoskeleton as mitochondria colocalized with actin cables in the retention zone, and mutations that perturb actin dynamics or actin-mitochondrial interactions produced retention defects. Our results support the model that equal distribution of mitochondria during cell division is a consequence of two actin-dependent processes: movement of some mitochondria into the daughter bud and immobilization of others in the mother cell.     

Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K-Akt signaling

Phosphoinositide-3 kinase (PI3K)/Akt signaling is activated by growth factors such as insulin and epidermal growth factor (EGF) and regulates several functions such as cell cycling, apoptosis, cell growth, and cell migration. Here, we find that Kank is an Akt substrate located downstream of PI3K and a 14-3-3-binding protein. The interaction between Kank and 14-3-3 is regulated by insulin and EGF and is mediated through phosphorylation of Kank by Akt. In NIH3T3 cells expressing Kank, the amount of actin stress fibers is reduced, and the coexpression of 14-3-3 disrupted this effect. Kank also inhibits insulin-induced cell migration via 14-3-3 binding. Furthermore, Kank inhibits insulin and active Akt-dependent activation of RhoA through binding to 14-3-3. Based on these findings, we hypothesize that Kank negatively regulates the formation of actin stress fibers and cell migration through the inhibition of RhoA activity, which is controlled by binding of Kank to 14-3-3 in PI3K-Akt signaling.     

ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPγS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPγS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion–like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH₂-terminally truncated Δ17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPγS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Δ17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.     

Effect of Rho-associated kinase inhibition on actin cytoskeleton structure and calcium response in glioma C6 cells

The role of actin cytoskeleton functional state in glioma C6 cell morphology and calcium signaling was investigated through modification of myosin II activity by blocking Rho-associated kinase with the specific inhibitor Y-27632. Treatment of glioma C6 cells with ROCK inhibitor resulted in actin cytoskeleton reorganization and also in the changed shape and distribution of mitochondria. Changes in the distribution of ER, the main calcium store in glioma C6 cells, were not visible. The inhibition of myosin II activity influences the first phase of calcium signaling evoked by agonist, and both phases of thapsigargin-evoked calcium response. We suggest that the observed increase in Ca2+ release from intracellular stores induced by IP3 formation as well as inhibition of SERCA ATPase is at least in part related to severely affected mitochondria. Enhancement of capacitative calcium entry evoked by thapsigargin is probably associated with the reorganization of the acto-myosin II system. ATP-induced calcium response presents no changes in the second phase. We observed that ATP stimulation of Y-27632 pretreated cells leads to immediate morphological rearrangement of glioma C6 cells. It is a consequence of actin cytoskeleton reorganization: formation of stress fibers and relocation of phosphorylated myosin II to actin filaments. It seems that the agonist-evoked strong calcium signal may be sufficient for myosin II activation and the stress fiber organization. This is the first work showing the dependence between the functional state of the acto-myosin II system and calcium signaling stressing the reversible character of this relationship.