The effects of cytochalasin B on the cytoplasmic contractile network revealed by whole-cell transmission electron microscopy

The ultrastructure of the contractile response to cytochalasin B (CB) has been studied using whole-cell electron microscopy. The actin-containing contractile network rapidly condenses into numerous stellate microfilament foci (SMF). These SMF punctuate the cytoplasm, and are frequently associated with an extensive persistent cytoskeleton containing microtubules and intermediate filaments. This association of SMF and persistent cytoskeleton appears to mediate the arborized morphology induced by CB. Eventually SMF aggregate and migrate towards the nucleus. Concomitantly the cell surface is differentiated into clusters of miniblebs which migrate to the nucleus. SMF loss from the periphery resulted in respreading to a flattened angular morphology within which the nucleus was frequently displaced. The role of the actin network, and the mechanism of these CB-induced contractile alterations are discussed.     

Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB)

The organization of microfilaments and microtubules in cultured cells before and after the addition of cytochalasin B (CB) was studied both by electron microscopy and immunofluorescence microscopy using antibodies specific for actin, tubulin and tropomyosin. CB induces a rapid disorganization of normal microfilament bundles. Star-like patches of actin and tropomyosin are visualized in immunofluorescence microscopy and dense aggregates of condensed microfilaments are seen in electron microscopy. The integrity of the microtubules is not changed by CB treatment. Addition of CB to glycerinated cells, in contrast to normal cells, does not result in the disorganization of microfilament bundles. CB-treated glycerinated models can still contract upon addition of ATP. Thus the CB-induced rearrangement of microfilament bundles occurs only in vivo and not in glycerinated cell contractility models.     

Disassembly of actin filaments by botulinum C2 toxin and actin-filament-disrupting agents induces assembly of microtubules in human leukaemia cell lines

BACKGROUND INFORMATION: C(2) toxin produced by Clostridium botulinum types C and D ADP-ribosylates actin monomers and inactivates their polymerization activities. The disassembly of actin filaments by C(2) toxin induces a polarization of cultured human leukaemia cell lines. RESULTS: The polarization induced by C(2) toxin was temperature dependent and was prevented by nocodazole, a microtubule-disrupting agent, whereas it was promoted by paclitaxel, a microtubule-stabilizing agent. The fluorescence staining of polarized cells indicated an increase in microtubule assembly accompanying disassembly of actin filaments. Furthermore, several actin-filament-disrupting agents, other than C(2) toxin, also induced microtubule assembly and cell polarization, irrespective of their different mechanisms of action. The effects induced by some of the agents, which have lower binding affinities for actin, were reversible in response to the re-assembly of actin filaments. CONCLUSIONS: Thus the disassembly of actin filaments by C(2) toxin and actin-filament-disrupting agents induces assembly of microtubules followed by polarization of human leukaemia cell lines, indicating that the assembly/disassembly equilibrium of actin filaments influences the dynamics of microtubules, which control cell morphology and, in turn, diverse cellular processes.     

The effect of the depolymerization and disintegration of the microtubular system on the cytoskeleton of untransformed and transformed cells]

How important are the changes of microtubule control for the realization of actin cortex changes during neoplastic transformation? To answer this question we studied the actin cytoskeleton and intermediate filaments condition after colcemid destruction or taxol disintegration of microtubule system in non-transformed cells BALB/c 3T3 and in the same cells transformed by Ha-ras gene. We have come to a conclusion that the differences between non-transformed and transformed cells in the actin cytoskeleton organization remain the same after specific inhibitor action on the microtubules; after the microtubules are destroyed the differences between the two cell types appear in the intermediate filament organization; there are reasons to assume that changes in the actin cortex structure may play the central role in morphological transformation expression.     

Immunofluorescence demonstration of tubulin and actin in estrogen-induced rat prolactinoma cells in vitro : Alteration of their distribution after bromocriptine, colchicine and cytochalasin B treatments

Cultured cells in vitro from estrogen-induced rat prolactin-secreting adenomas (prolactinomas) were examined by indirect immunofluorescence microscopy for the distribution of cytoskeletal proteins and alterations of cytoskeleton after treatment with bromocriptine, colchicine and cytochalasin B (CB). After 8 days in culture, prolactinoma cells were well expanded and developed cytoplasmic processes were seen. The cytoplasmic microtubules were observed as fine reticular networks radiating from perinuclear portions toward the cell periphery when decorated with an antibody against tubulin. On the other hand, the actin filaments showed diffuse and spotty distribution when detected with an anti-actin antibody. Contaminated fibroblasts showed a reticular distribution of microtubules and a parallel array of actin cables which corresponds to "stress fibers" throughout the cytoplasm. After treatment with bromocriptine, the reticular distribution of microtubules in prolactinoma cells changed into a coarse and sparse pattern, which was identical with the changes in the distribution of tubulin after treatment with colchicine. On the other hand, distribution of actin was not affected by bromocriptine. Bromocriptine treatment did not alter the distribution of microtubules and actin filaments in fibroblasts, whereas colchicine changed the distribution of microtubules in both prolactinoma cells and fibroblasts. CB treatment changed the localization of actin filaments in both kinds of cells. These in vitro studies indicated bromocriptine would selectively affect the cytoplasmic microtubular system of prolactinoma cells.     

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei.     

Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores

The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletal morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule-inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha-melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus-end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies.     

Cytochalasin B-induced redistribution of cytokeratin filaments in PtK1 cells

Indirect immunofluorescence demonstrated a dramatic reorganization of cytokeratin filaments produced by cytochalasin B (CB) treatment of PtK1 cells. Much of the normal cytokeratin network became arranged into a latticework consisting of bundles of cytokeratin filaments that radiated from, and interconnected, distinct foci. Electron microscopy showed foci to be dense granular regions through which bundles of cytokeratin filaments looped. Composition of the foci included actin, myosin, and alpha-actinin, as shown by labeling with rhodamine phalloidin or specific antisera. Simultaneous treatment with CB and colchicine was not required for lattice formation, but did produce more extensive development than did CB alone. In cells treated only with CB, the microtubule network remained intact, even in regions of extensive lattice formation. These results contrast sharply with those of Knapp et al (J. Cell Biol. 97:1788 [1983b]), who found lattice formation dependent upon simultaneous CB and colchicine treatment. Time-course and dose-response studies of CB treatment showed lattice formation to follow disruption of stress fibers and the concentration of actin into distinct patches that marked the location of lattice foci. Overall results suggest a structural association between microfilaments and cytokeratin filaments that produces the lattice pattern upon CB-induced disruption of stress fibers. Lattice formation was not limited to a specific cell-cycle stage, since G1, G2, and M cells displayed the lattice. Treatment of cells with dihydro-CB and experiments with enucleated cells showed that lattice formation was dependent upon neither the inhibition of sugar transport nor the nuclear extrusion effects of CB.     

Roles of microfilaments and microtubules in paxillin dynamics

We investigated the roles of microfilaments and microtubules in the localization and tyrosine phosphorylation of paxillin, a focal adhesion-associated signaling molecule, in bovine aortic endothelial cells (BAECs). Paxillin tyrosine phosphorylation is inhibited by cytochalasin D (CD), but slightly increased by colchicine and paclitaxol (taxol). CD also caused an overall disassembly of paxillin-containing focal adhesions (paxillin-FAs) and translocation of paxillin to the cytoplasm and perinuclear region with a diffuse distribution. Meanwhile, colchicine and taxol caused a disassembly of paxillin-FAs from cell periphery and lamellipodia, and their assembly in cell center. These results indicate that actin filaments are important in paxillin assembly in the FAs of the whole ECs and that microtubules are critical in paxillin assembly in cell periphery and lamellipodia; thus the microfilaments and microtubules play differential roles in the dynamics of paxillin assembly/disassembly. Our findings also suggest that tyrosine phosphorylation is an important element in paxillin dynamics at FAs.     

Heat shock induction of intranuclear actin rods in cultured mammalian cells

Incubation of cultured cells of mouse C3H-2K fibroblastic cell line and other mammalian cell lines at 42.0-43.0 degrees C for 30 min or longer caused disintegration of normal actin structures including stress fibers, and induced formation of intranuclear actin paracrystal-like structures, called actin rods. When cells exposed to the elevated temperatures were shifted back to 37 degrees C, normal actin structures were regained. Pretreatment of cells at moderately high temperatures such as 38.5 degrees C inhibited formation of the actin rods upon subsequent exposure to 42.0 degrees C. Neither microtubules nor intermediate filaments were disrupted by the heat treatment. Several heat shock proteins were found to be synthesized under the conditions where actin rods were induced. However, there is no causal relationship between two cellular events, the induction of intranuclear actin rods and the synthesis of heat shock proteins.     

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: I. The effect of cadmium

Summary. The aim of the study was to elucidate the effect of cadmium ions on the arrangement of the actin and tubulin cytoskeleton, as well as the relationships between cytoskeletal changes and growth processes in the green filamentous alga Spirogyra decimina. Batch cultures of algae were carried out under defined conditions in the presence of various cadmium concentrations. In control cells, the cytoskeleton appeared to be a transversely oriented pattern of both microtubules and actin filaments of various thickness in the cell cortex; colocalization of cortical microtubules and actin filaments was apparent. Microtubules were very sensitive to the presence of cadmium ions. Depending on the cadmium concentration and the time of exposure, microtubules disintegrated into short rod-shaped fragments or they completely disappeared. A steep increase in cell width and a decrease in growth rate accompanied (and probably ensued) a very rapid disintegration of microtubules. Actin filaments were more stable because they were disturbed several hours later than microtubules at any cadmium concentration used. When cadmium ions were washed out, the actin cytoskeleton was rebuilt even in cells in which actin filaments were completely disintegrated at higher cadmium concentrations (40 or 100 μM). The much more sensitive microtubules were regenerated after treatment with lower cadmium concentrations (10 or 15 μM) only. Correspondence and reprints: Centre of Phycology, Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, 379 82 Třeboň, Czech Republic.     

Cortical Actin Filaments Potentially Interact with Cortical Microtubules in Regulating Polarity of Cell Expansion in Primary Roots of Maize (Zea mays L.)

Evidence is accumulating implicating cortical microtubules in the directional control of cell expansion. However, the role of actin filaments in this process is still uncertain. To determine the involvement of actin in cell elongation, the organization of actin filaments in primary roots of maize (Zea mays L.) was examined by use of an improved fluorochrome-conjugated phalloidin-labeling method. With this method, a previously undetected state of actin organization was revealed in the elongation and maturation zone of maize roots. Fine transversely oriented cortical actin was observed in all cells of the elongation zone, including the epidermis, cortex, and vascular tissues. The orientation of cortical actin shifted from a predominantly transverse orientation to oblique, longitudinal, and/or random arrangements as the cells matured. The reorientation of cortical actin in maturing root cells mimics the behavior of cortical microtubules reported in other studies. Furthermore, roots treated with the microtubule-stabilizing drug taxol improved the quality of actin preservation as evidenced by the thicker bundles of cortical actin. This suggested that taxol was also capable of stabilizing the cortical actin networks. The elongation of roots exposed to 1 micromole Latrunculin B, an actin-disrupting drug, was inhibited, and after 24 h the roots exhibited moderate swelling particularly along the elongation zone. Latrunculin B also caused microtubules to reorient from transverse to oblique arrays. The results from this study provide evidence that cortical microtubules and actin filaments respond in a coordinated way to environmental signals and may well depend on both elements of the cytoskeleton.     

Secondary lysosomes as an integral part of the cytoskeleton: a morphological study in rat Kupffer cells

Rat Kupffer cells contain the three major cytoskeletal components: microfilaments (MF), microtubules (MT), and intermediate filaments (IF) of the vimentin type. Previous cytomagnetometric data obtained from alveolar macrophages and rat Kupffer cells in culture provided evidence that actin filaments contribute to the movements of lysosomes. The lysosomal transport in living cells was affected, when the MFs were selectively disturbed, whereas the depolymerization of the MTs had no effect on the lysosomal movement measured by cytomagnetometric means. Immunofluorescence and ultrastructural studies of isolated and cultured rat Kupffer cells, presented in this paper, will investigate the relationship between lysosomes and the cytoskeleton. The principal filamentous structure in the peripheral cytoplasm of Kupffer cells in a dense meshwork of actin filaments. The dimension of the meshes combined with the dimensions of lysosomes implies the necessity of either (i) disintegration of the actin filament cross-links, (ii) depolarymerization and redistribution of MF's, or (iii) a displacement of actin filaments by the lysosomes during the organelle transport. The presence of microtubules in cytoplasmic protrusions and their track from the periphery to the perinuclear region during interphase might play a role in the transport mechanism of lysosomes, the more so because microtubules could often be demonstrated in closest association with lysosomes even in the first phase of endocytosis. The distribution pattern of vimentin, found as a dense interconnected framework surrounding the lysosomes like a basket, could play a role in positioning the organelles. The dynamic functions of MF's and MT's and their multifunctionality led to an adaptive and flexible organization of these filaments which may both be involved in lysosomal motion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of actin filaments and microtubules to quasi-in situ tensile properties and internal force balance of cultured smooth muscle cells on a substrate

The effects of actin filaments (AFs) and microtubules (MTs) on quasi-in situ tensile properties and intracellular force balance were studied in cultured rat aortic smooth muscle cells (SMCs). A SMC cultured on substrates was held using a pair of micropipettes, gradually detached from the substrate while maintaining in situ cell shape and cytoskeletal integrity, and then stretched up to approximately 15% and unloaded three times at the rate of 1 mum every 5 s. Cell stiffness was approximately 20 nN per percent strain in the untreated case and decreased by approximately 65% and approximately 30% following AF and MT disruption, respectively. MT augmentation did not affect cell stiffness significantly. The roles of AFs and MTs in resisting cell stretching and shortening were assessed using the area retraction of the cell upon noninvasive detachment from thermoresponsive gelatin-coated dishes. The retraction was approximately 40% in untreated cells, while in AF-disrupted cells it was <20%. The retraction increased by approximately 50% and decreased by approximately 30% following MT disruption and augmentation, respectively, suggesting that MTs resist intercellular tension generated by AFs. Three-dimensional measurements of cell morphology using confocal microscopy revealed that the cell volume remained unchanged following drug treatment. A concomitant increase in cell height and decrease in cell area was observed following AF disruption and MT augmentation. In contrast, MT disruption significantly reduced the cell height. These results indicate that both AFs and MTs play crucial roles in maintaining whole cell mechanical properties of SMCs, and that while AFs act as an internal tension generator, MTs act as a tension reducer, and these contribute to intracellular force balance three dimensionally.     

Effects of microtubule disruption on force, velocity, stiffness and [Ca(2+)](i) in porcine coronary arteries

Force generated by smooth muscle cells is believed to result from the interaction of actin and myosin filaments and is regulated through phosphorylation of the myosin regulatory light chain (LC(20)). The role of other cytoskeleton filaments, such as microtubules and intermediate filaments, in determining the mechanical output of smooth muscle is unclear. In cultured fibroblasts, microtubule disruption results in large increases in force similar to contractions associated with LC(20) phosphorylation (15). One hypothesis, the "tensegrity" or "push-pull" model, attributes this increase in force to the disruption of microtubules functioning as rigid struts to resist force generated by actin-myosin interaction (9). In porcine coronary arteries, the disruption of microtubules by nocodazole (11 microM) also elicited moderate but significant increases in isometric force (10-40% of a KCl contracture), which could be blocked or reversed by taxol (a microtubule stabilizer). We tested whether this nocodazole-induced force was accompanied by changes in coronary artery stiffness or unloaded shortening velocity, parameters likely to be highly sensitive to microtubule resistance elements. Few changes were seen, ruling out push-pull mechanisms for the increase in force by nocodazole. In contrast, the intracellular calcium concentration, measured by fura 2 in the intact artery, was increased by nocodazole in parallel with force, and this was inhibited and/or reversed by taxol. Our results indicate that microtubules do not significantly contribute to vascular smooth muscle mechanical characteristics but, importantly, may play a role in modulation of Ca(2+) signal transduction.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.     

Uterine smooth muscle cells in primary culture

Summary Smooth muscle cells (SMC) were enzymatically isolated from the myometrium of adult rat and human uteri and grown in primary culture. Cell fine structure and cytoskeletal organization were followed by transmission electron microscopy and cytochemical demonstration of actin filaments, microtubules and intermediate filaments, and initiation of DNA synthesis was investigated by thymidine autoradiography. During the first few days in culture the cells spread out on the substrate and went through a morphological transformation including loss of myofilaments followed by formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Actin filaments aggregated in stress fibers spanning the entire length of the cells and microtubules and intermediate filaments formed a radiating system originating in the juxtanuclear region. In vivo, the SMC contained intermediate filaments reactive for desmin, but as early as the first day of culture expressed vimentin as well. For five days at least, all cells remained positive for both proteins, but the staining for desmin decreased while that for vimentin increased. This structural modification was accompanied by initiation of DNA synthesis, with a peak on day 3 (45–55% labeled nuclei). Subconfluent, growth-arrested primary cultures responded weakly to purified platelet-derived growth factor and serum, and in secondary cultures no response to the mitogenic stimulation was obtained. The observations indicate that uterine SMC cultivated in vitro undergo a transformation from contractile to synthetic phenotype, similar to the transformation described previously for arterial SMC under the same conditions. The proliferative potential of the uterine cells is, however, markedly lower. The findings support the notions that the transition into synthetic phenotype is a necessary but not sufficient requirement for initiation of DNA synthesis in SMC and that visceral and vascular SMC represent separate differentiation pathways.     

Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins

We have used low shear viscometry and electron microscopy to study the interaction between pure actin filaments and microtubules. Mixtures of microtubules having microtubule-associated proteins (MAPs) with actin filament have very high viscosities compared with the viscosities of the separate components. MAPs themselves also cause a large increase in the viscosity of actin filaments. In contrast, mixtures of actin filaments with tubulin polymers lacking MAPs have low viscosities, close to the sum of the viscosities of the separate components. Our interpretation of these observations is that there is an interaction between actin filaments and microtubules which requires MAPs. This interaction is inhibited by ATP and some related compounds. Electron micrographs of thin sections through mixtures of actin and microtubules show numerous close associations between the two polymers which may be responsible for their high viscosity.     

Phorbol 12-myristate 13-acetate prevents isoproterenol-induced morphological change in cultured vascular smooth muscle cells

The effect of phorbol 12-myristate 13-acetate (PMA) on isoproterenol (ISO)- and dibutyryl cAMP (dBcAMP)-induced morphological change and cytoskeletal reorganization was studied in cultured vascular smooth muscle cells (VSMC) using the fluorescence staining of actin and microtubules. The treatment of VSMC with 1.0 μM of ISO or with 1.0 mM of dBcAMP for 90 min induced the disruption of actin-containing stress fibers followed by cytoplasmic arborization. The addition of 100 or 10 nM of PMA prevented both the destruction of actin fibers and cell arborization induced either by ISO or by dBcAMP. However, PMA rather enhanced cAMP production stimulated by ISO. I-Oleoyl-2-acetylsn-glycerol (100 μg/ml) mimicked this inhibitory effect of PMA whereas 4a-phorbol 12,13-didecanoate (100 nM) failed to block the arborization. These results indicated that the inhibition of arborization by PMA was mediated through the activation of protein kinase C. Colchicine at 5.0 μM also had an inhibitory effect on ISO- and dBcAMP-induced cell arborization. However, immunofluorescence studies revealed that colchicine but not PMA elicited the reorganization of microtubules, suggesting that the effect of PMA was mediated through a mechanism different from that of colchicine. These observations indicated that the morphology of VSMC was regulated through the alteration of cytoskeletal organization induced by cAMP-mediated and by protein kinase C-dependent systems.     

Regulation of stretch-activated intracellular calcium transients by actin filaments.

Stretch activation of cation-permeable channels may be an important proximal sensory mechanism in mechanotransduction. As actin filaments may mediate cellular responses to changes of the mechanical properties of the substrate and regulate stretch-induced calcium transients, we examined the role of actin filaments and substrate flexibility in modulating the amplitude of stretch-activated intracellular calcium transients. Human gingival fibroblasts were subjected to mechanical stretch through integrins by magnetic force acting on collagen-coated ferric oxide beads. Intracellular calcium concentration was measured in fura-2-loaded cells by ratio fluorimetry. Cytochalasin D-treatment greatly increased (3-fold) the amplitude of stretch-activated calcium transients in well-spread cells grown on glass coverslips while phalloidin, colchicine or taxol exerted no signficant effects, indicating that actin filaments but not microtubules modulate stretch-activated calcium transients. In freshly plated cells with rounded shapes and poorly developed cortical actin filaments, stretch-induced calcium transients were of 3-fold higher amplitude than well-spread cells plated for 6-24 hrs and with well developed actin filaments. Cells plated on soft collagen-polyacrylamide gels showed round morphology but exhibited <50% of the response to stretch of well-spread cells on inflexible gels. Notably, cells on soft gels showed very heavy phalloidin staining for cortical actin filaments compared with cells on more inflexible surfaces which showed only light staining for cortical actin. While cell shape may have some effect on responsiveness to mechanical stretch, the rigidity of the cell membrane mediated by the extensive cortical actin network appears to be a central determinant in the regulation of stretch-induced calcium signals.