Cytochalasin D induces increased actin synthesis in HEp-2 cells.

In HEp-2 cells treated with 0.2 to 2.0 microM cytochalasin D (CD) for 7.5 to 24 h there was a 20 to 50% relative increase in actin content (units of actin per microgram of total cell protein). This augmentation, which was concentration and time dependent, was prevented by treatment with cycloheximide during exposure to CD. A 15 to 20% increase in the relative rate of actin synthesis in CD-treated HEp-2 cells (0.2 to 2.0 microM CD) was detectable after 1 h of treatment and increased to 30 to 50% by 24 h. This increased rate of actin synthesis was apparently responsible for the higher actin content of CD-treated HEp-2 cells. The concentration dependence of these effects of CD on actin metabolism correlated with the pattern seen for CD-triggered changes in cellular morphology and the underlying rearrangements of the actin-containing cytoskeletal structures, suggesting that the effects on metabolism and morphology were interrelated. Since the rapidly occurring cytoskeletal reorganization preceded the effects of CD on actin metabolism, it is proposed that actin synthesis is induced by the cytoskeletal rearrangement resulting from exposure to CD.     

Evidence for regulation of actin synthesis in cytochalasin D-treated HEp-2 cells

In HEp-2 cells treated with 0.2 or 2.0 μM cytochalasin D (CD), the relative rate of actin synthesis increased for about 12 h and then reached a plateau; this increase was suppressed by actinomycin D (AD). When CD was washed from cells which had been treated for 20 h, the elevated rate of actin synthesis declined to the control value within ca 4 h, as the actin-containing cytoskeletal components rearranged by CD recovered their normal morphology. Subsequently, actin synthesis was depressed below control values for a prolonged period; during recovery from 2 h treatment with CD, this depression was of much shorter duration. Re-addition of CD to cells after a 3 h recovery period again induced the cytoskeletal alterations characteristic of CD treatment but did not reverse the prior decline in the rate of actin synthesis. In HEp-2 cells treated with cycloheximide during exposure to CD for 20 h, the relative rate of actin synthesis measured after removal of cycloheximide was twofold higher than with CD alone and such cells exhibited a twofold slower decline in the rate of actin synthesis during recovery from CD in the continued presence of cycloheximide. These effects of cycloheximide, which resemble observations on “super-induction”, suggest that actin synthesis in CD-treated and recovering HEp-2 cells may be regulated by a repressor protein. The possibility that the proposed repressor protein is actin and that actin may thus be a feedback inhibitor of its own synthesis is discussed.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

Cytochalasin D alters the rate of synthesis of some HEp-2 cytoskeletal proteins. Examination by two-dimensional gel electrophoresis

The most abundant proteins of HEp-2 cells were resolved by two-dimensional gel electrophoresis. The protein spots corresponding to several cytoskeletal proteins (vimentin, alpha-tubulin, beta-tubulin, alpha-actinin, tropomyosins, and cytokeratins) were identified by comigration with protein markers or by immunological techniques. After treatment of HEp-2 cells with 0.2 microM or 2.0 microM cytochalasin D for 20 h, radioautograms of two-dimensional gel patterns of lysates from cells pulse-labeled with [35S]methionine indicated that the drug altered the rate of synthesis of some proteins. The relative rate of synthesis of the identified cytoskeletal proteins was measured. Synthesis of alpha-actinin, the higher-molecular-mass pair of tropomyosins and actin were similarly increased with cytochalasin D treatment, suggesting coordinate induction. Vimentin and tubulin synthesis was depressed. One cytokeratin exhibited an increase in synthesis comparable to actin, another was increased to a lesser extent and one was decreased.     

Bacterial lipopolysaccharide induces cytoskeletal rearrangement in small intestinal lamina propria fibroblasts: actin assembly is essential for lipopolysaccharide signaling

Cytoskeletal proteins are major components of the cell backbone and regulate cell shape and function. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) on the dynamics and organization of the cytoskeletal proteins, actin, vimentin, tubulin and vinculin in human small intestinal lamina propria fibroblasts (HSILPF). A noticeable change in the actin architecture was observed after 30 min incubation with LPS with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 2 h. Reorganization of the vimentin network into vimentin bundling was conspicuous at 2 h. With further increase in the time period of LPS exposure, diffused staining of vimentin along with vimentin bundling was observed. Vinculin plaques distributed in the cell body and cell periphery in the control cells rearrange to cell periphery in LPS-treated cells by 30 min of LPS exposure. However, there was no change in the tubulin architecture in HSILPF in response to LPS. LPS increased the F-actin pool in HSILPF in a concentration-dependent manner with no difference in the level of G-actin. A time-dependent study depicted an increase in the G-actin pool at 10 and 20 min of LPS exposure followed by a decrease at further time intervals. The F-actin pool in LPS-treated cells was lower than the control levels at 10 and 20 min of LPS exposure followed by a sharp increase until 120 min and finally returning to the basal level at 140 and 160 min. Further (35)S-methionine incorporation studies suggested a new pool of actin synthesis, whereas the synthesis of other cytoskeletal filaments was not altered. Cytochalasin B, an actin-disrupting agent, severely affected the LPS induced increased percentage of 'S' phase cells and IL-6 synthesis in HSILPF. We conclude that dynamic and orchestrated organization of the cytoskeletal filaments and actin assembly in response to LPS may be a prime requirement for the LPS induced increase in percentage of 'S' phase cells and IL-6 synthesis     

Distinct effects of calcium- and cyclic AMP-enhancing factors on cytoskeletal synthesis and assembly in mouse osteoblastic cells.

Previous studies have indicated that the effects of parathyroid hormone (PTH) on osteoblastic function involve alteration of cytoskeletal assembly. We have reported that after a transitory cell retraction, PTH induces respreading with stimulation of actin, vimentin and tubulins synthesis in mouse bone cells and that this effect is not mediated by cAMP. In order to further elucidate the role of intracellular cAMP and calcium on PTH action on bone cell shape and cytoskeleton we have compared the effects of calcium- and cAMP-enhancing factors on actin, tubulin and vimentin synthesis in relation with mouse bone cell morphology, DNA synthesis and alkaline phosphatase activity as a marker of differentiation. Confluent mouse osteoblastic cells were treated with 0.1 mM isobutylmethylxanthine (IBMX) for 24 h. This treatment caused an increase in the levels of cytoskeletal subunits associated with an elevation of cAMP. Under these conditions, PTH (20 nM) and forskolin (0.1 microM) produced persistent cytoplasmic retraction. PTH and forskolin treatment in presence of IBMX (24 h) induced inhibitory effects on actin and tubulin synthesis evaluated by [35S]methionine incorporation into cytoskeletal proteins identified on two-dimensional gel electrophoresis. Under these culture conditions PTH and forskolin also caused disassembly of microfilament and microtubules as shown by the marked reduction in Triton X soluble-actin and alpha- and beta-tubulins. In contrast, incubation of mouse bone cells with 1 microM calcium ionophore A23187 (24 h) resulted in increased monomeric and polymeric forms of actin and tubulin while not affecting intracellular cAMP. Alkaline phosphatase activity was increased in all conditions while DNA synthesis evaluated by [3H]thymidine incorporation into DNA was stimulated by PTH combined with forskolin and inhibited by the calcium ionophore. These data indicate that persistent elevation of cAMP levels induced by PTH and forskolin with IBMX cause cell retraction with actin and tubulin disassembly whereas rising cell calcium induces cytoskeletal protein assembly and synthesis in mouse osteoblasts. The results point to a distinct involvement of calcium and cAMP in both cytoskeletal assembly and DNA synthesis in mouse bone cells.     

The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C

The control of cytoskeletal actin and exocytosis was examined in intact and digitonin-permeabilized chromaffin cells. Cytoskeletal actin was assayed by determining the actin content of Triton-insoluble cytoskeletons. The secretagogues nicotine, high K+ and Ba2+ resulted in a rapid reduction in the amount of actin associated with the cytoskeleton. The effect of nicotine but not high K+ on cytoskeletal actin was independent of external Ca2+ and the reduction in cytoskeletal actin was mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate suggesting a role for protein kinase C. In digitonin-permeabilized cells micromolar calcium produced both catecholamine secretion and a reduction in cytoskeletal actin. The reduction in cytoskeletal actin was transient. Secretion was enhanced by the GTP analogue guanosine 5'-(3-O-thio)triphosphate and the analogue also reduced cytoskeletal actin at low calcium levels. The effects of guanosine 5'-(3-O-thio)triphosphate were inhibited by the phospholipase C inhibitor neomycin and were mimicked by 12-O-tetradecanoylphorbol-13-acetate. An additional GTP analogue, guanyl-5'-yl imidodiphosphate, had no effect on cytoskeletal actin. These results provide further evidence for a requirement for reorganisation of cortical actin in the secretory processes and suggest that the reduction in actin associated with the cytoskeleton may be mediated by protein kinase C and/or calcium in intact and permeabilized chromaffin cells.     

In vitro regulation of granulosa cell differentiation. Involvement of cytoskeletal protein expression

The link between the biochemical and morphological differentiation of granulosa cells was studied by investigating the organization and the expression of cytoskeletal proteins which determine cell shape and contacts. In cells treated with follicle-stimulating hormone (FSH), in a serum- and growth factor-free medium, or with other compounds which elevate cellular cAMP levels, the synthesis of the adherens junction proteins, vinculin, alpha-actinin, and actin was reduced significantly when compared to unstimulated cells (7-fold for vinculin, 5-fold for alpha-actinin, and 3-fold for actin). The in vitro translatability of the mRNAs coding for these proteins and the level of actin mRNA determined by RNA blot hybridization were generally reduced in differentiating cells. The synthesis and the organization of vimentin and tubulin was unaffected during this process, whereas the organization of actin and vinculin was dramatically affected, with FSH-treated cells displaying a diffuse pattern of actin and vinculin, with very little vinculin in adhesion plaques. Gonadotropin-releasing hormone agonist and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate which are known to antagonize the cAMP-mediated biochemical differentiation of granulosa cells by reducing cAMP levels or by activating protein kinase C and phospholipid turnover, blocked to a large extent the FSH-induced effect on the adherens junction proteins. Epidermal growth factor, which blocked the FSH-induced cAMP increase, but not the FSH-induced progesterone production, failed to block the synthesis of vinculin, alpha-actinin, and actin. Cytochalasin B could induce steroidogenesis and similar changes in the synthesis of these cytoskeletal proteins, whereas fibronectin, which causes cell spreading, blocked in part the FSH-induced effect on the expression of cytoskeletal proteins. The modulation of cytoskeletal proteins may therefore be an essential feature of programmed differentiation events leading to the final phenotype of granulosa cells.     

Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure

We studied the association of several eucaryotic viral and cellular mRNAs with cytoskeletal fractions derived from normal and virus-infected cells. We found that all mRNAs appear to associate with the cytoskeletal structure during protein synthesis, irrespective of their 5' and 3' terminal structures: e.g., poliovirus that lacks a 5' cap structure or reovirus and histone mRNAs that lack a 3' poly A tail associated with the cytoskeletal framework to the same extent as capped, polyadenylated actin mRNA. Cellular (actin) and viral (vesicular stomatitis virus and reovirus) mRNAs were released from the cytoskeletal framework and their translation was inhibited when cells were infected with poliovirus. In contrast, actin mRNA was not released from the cytoskeleton during vesicular stomatitis virus infection although actin synthesis was inhibited. In addition, several other conditions under which protein synthesis is inhibited did not result in the release of mRNAs from the cytoskeletal framework. We conclude that the association of mRNA with the cytoskeletal framework is required but is not sufficient for protein synthesis in eucaryotes. Furthermore, the shut-off of host protein synthesis during poliovirus infection and not vesicular stomatitis virus infection occurs by a unique mechanism that leads to the release of host mRNAs from the cytoskeleton.     

Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules.

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.     

Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis

Hyperthermia induces several cellular responses leading to morphological changes, cell detachment and death. Loss of integrins from the cell surface after acute heat-treatment may block several physiological signalling pathways, but whether the assembly network between integrin and cytoskeletal actin is perturbed during hyperthermic treatment is unknown. In this study we tested this hypothesis by evaluating cell morphology, protein cytoskeletal profile and integrin CD11a content in both adherent and floating SK-N-MC human neuroblastoma cells. Morphological and cytometric analyses confirmed that hyperthermia is an effective apoptotic trigger, revealing the typical chromatin margination, cell shape changes and 7-AAD incorporation. After hyperthermia, cytoskeletal proteins showed an increase of high-molecular-weight aggregates and a significant decrease of both actin and CD11a content with respect to control cells. The integrin CD11a and membrane-bound actin alterations found in detached floating neuroblastoma cells recovered after heat-shock may cause the cytoskeletal abnormalities related to the observed surface cell rounding/blebbing and anoikis, early events of hyperthermia-induced programmed cell death.     

Increase in cytoskeletal actin induced by inositol 1,4-bisphosphate in saponin-permeated pig platelets

Inositol 1,4-bisphosphate (IP₂), which rapidly accumulates during cell activation, strongly stimulates an increase in cytoskeletal actin in saponin-permeated platelets, and the effect is insensitive to 5′-Chloro-5′-deoxyadenosine. Within 10 s, the amount of cytoskeletal actin in platelets rapidly increases by 41%, and then slowly increases further. IP₂ induces the increase in cytoskeletal actin in a dose-dependent manner. The half-maximal effect requires approximately 2 μM of IP₂ Inositol 1,4,5- triphosphate, the messenger for Ca²⁺ release, causes the increase in cytoskeletal actin, but is less effective than IP₂. Inositol 1-monophosphate and inositol 2-monophosphate have no effect on cytoskeletal actin. Phorbol 12-myristate 13-acetate, which has been shown to activate IP₃ 5′-phosphatase through protein kinase C, stimulates the increase in cytoskeletal actin. Spermine, an inhibitor of IP₃ 5′-phosphatase, inhibits the thrombin stimulated increase in cytoskeletal actin. These results suggest that IP₂ may be a messenger that controls the organization of actin filaments during cell activation. This study presents the first evidence for IP₂ as a messenger during cell activation.     

Quantitative analysis of cytoskeletal proteins throughout the cell cycle of the MRC-5 fibroblastic cell line

Fluorescent dyes were used to stain actin, vimentin, tubulin and DNA in the same MRC-5 fibroblastic cells. Cytofluorometry and image analysis were then used to quantitatively evaluate the F actin, vimentin and tubulin content throughout the cell cycle. The results showed that different cells can have the same DNA content while their cytoskeletal protein content is variable. The data also showed that cytoskeletal protein content variations exist throughout the cell cycle of the fibroblastic cell line. The F actin content increased during the cell cycle from G1 to G2 phases and decreased in M phase. The amount of tubulin in the G2 was about twice as much as that in the G1 phase, before decreasing in the M phase; there was a threshold of tubulin content for G2 cells entering S phase.     

Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration

Injury to the axons of facial motoneurons stimulates increases in the synthesis of actin, tubulins, and GAP-43, and decreases in the synthesis of neurofilament proteins: mRNA levels change correspondingly. In contrast to this robust response of peripheral neurons to axotomy, injured central nervous system neurons show either an attenuated response that is subsequently aborted (rubrospinal neurons) or overall decreases in cytoskeletal protein mRNA expression (corticospinal and retinal ganglion neurons). There is evidence that these changes in synthesis are regulated by a variety of factors, including loss of endoneurially or target-derived trophic factors, positive signals arising from the site of injury, changes in the intraaxonal turnover of proteins, and substitution of target-derived trophic support by factors produced by glial cells. It is concluded that there is, as yet, no coherent explanation for the upregulation or downregulation of any of the cytoskeletal proteins following axotomy or during regeneration. In considering the relevance of these changes in cytoskeletal protein synthesis to regeneration, it is emphasized that they are unlikely to be involved in the initial outgrowth of the injured axons, both because transit times between cell body and injury site are too long, and because sprouting can occur in isolated axons. Injuryinduced acceleration of the axonal transport of tubulin and actin in the proximal axon is likely to be more important in providing the cytoskeletal protein required for initial axonal outgrowth. Subsequently, the increased synthesis and transport velocity for actin and tubulin increase the delivery of these proteins to support the increased volume of the maturing regenerating axons. Reduction in neurofilament synthesis and changes in neurofilament phosphorylation may permit the increased transport velocity of the other cytoskeletal proteins. There is little direct evidence that alterations in cytoskeletal protein synthesis are necessary for successful regeneration, nor are they sufficient in the absence of a supportive environment. Nevertheless, the correlation that exists between a robust cell body response and successful regeneration suggests that an understanding of the regulation of cytoskeletal protein synthesis following axon injury must be a part of any successful strategy to improve the regenerative capacity of the central nervous system.     

Cytoarchitecture of Kirsten sarcoma virus-transformed rat kidney fibroblasts: butyrate-induced reorganization within the actin microfilament network

Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading.     

Pinocytosis in Dictyostelium discoideum cells : A possible implication of cytoskeletal actin for pinocytotic activity

Pinocytosis in Dictyostelium discoideum axenic strain (Ax-2) cells in the growth phase is progressively inhibited at higher Ca2+ concentrations, the activity being maximal at submicromolar Ca2+ concentrations. The cytoskeletal actin content is also markedly reduced in the presence of 10 mM EGTA. This was confirmed by electronmicroscopy using intact cells and Triton X-100-insoluble cell cortices. Interestingly, the pinocytotic activity seemed to be somewhat increased in response to cytochalasin B (CB). Aggregation-competent Ax-2 cells which are usually devoid of pinocytotic activity can resume their activity considerably following treatment with 10 mM EGTA. Under these conditions, cytoskeletal actin declines markedly, as also was the case for growing Ax-2 cells. Our findings indicate a correlation between the pinocytotic activity and presence of cytoskeletal actin: reduced amounts of actin in the cell cortex seem to favour pinocytosis. Conceivably, membrane-associated actin filaments may function as a powerful anchor, restricting the flexibility of the cell membrane and thereby inhibiting the pinosome formation. Other properties of pinocytosis like a developmental change as well as the effects of pH and temperature are also described and were compared with the properties of wild-type strain, NC-4.     

Changes in the association of actin-binding proteins with the actin cytoskeleton during chemotactic stimulation of Dictyostelium discoideum

Triton-insoluble cytoskeletons were isolated from Dictyostelium discoideum AX3 cells prior to and following stimulation with 2'deoxy cyclic adenosine monophosphate (cAMP). Temporal changes in the content of actin and a 120,000 dalton actin-binding protein (ABP-120) in cytoskeletons following stimulation were monitored. Both actin and ABP-120 were incorporated into the cytoskeleton at 30-40 seconds following stimulation, which is cotemporal with the onset of pseudopod extension during stimulation of amoebae with chemoattractants. Changes in the content of total cytoskeletal protein and cytoskeletal myosin were determined under the same experimental conditions as controls. These proteins exhibited different kinetics from those of cytoskeletal ABP-120 and actin following the addition of 2'deoxy cAMP. The authors concluded that the association of ABP-120 with the cytoskeleton is regulated during cAMP signalling. Furthermore, these results indicate that ABP-120 is involved in cross-linking newly assembled actin filaments into the cytoskeleton during chemoattractant-stimulated pseudopod extension.     

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).     

Association of alphaB-crystallin, a small heat shock protein, with actin: role in modulating actin filament dynamics in vivo

Disruption of cytoskeletal assembly is one of the early effects of any stress that can ultimately lead to cell death. Stabilization of cytoskeletal assembly, therefore, is a critical event that regulates cell survival under stress. alphaB-crystallin, a small heat shock protein, has been shown to associate with cytoskeletal proteins under normal and stress conditions. Earlier reports suggest that alphaB-crystallin could prevent stress-induced aggregation of actin in vitro. However, the molecular mechanisms by which alphaB-crystallin stabilizes actin filaments in vivo are not known. Using the H9C2 rat cardiomyoblast cell line as a model system, we show that upon heat stress, alphaB-crystallin preferentially partitions from the soluble cytosolic fraction to the insoluble cytoskeletal protein-rich fraction. Confocal microscopic analysis shows that alphaB-crystallin associates with actin filaments during heat stress and the extent of association increases with time. Further, immunoprecipitation experiments show that alphaB-crystallin interacts directly with actin. Treatment of heat-stressed H9C2 cells with the actin depolymerzing agent, cytochalasin B, failed to disorganize actin. We show that this association of alphaB-crystallin with actin is dependent on its phosphorylation status, as treatment of cells with MAPK inhibitors SB202190 or PD98059 results in abrogation of this association. Our results indicate that alphaB-crystallin regulates actin filament dynamics in vivo and protects cells from stress-induced death. Further, our studies suggest that the association of alphaB-crystallin with actin helps maintenance of pinocytosis, a physiological function essential for survival of cells.