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.     

Cytochalasin D-induced increase in actin synthesis and content in a variety of cell types

Treatment of a variety of mesenchymal cells (normal and transformed rat fibroblasts, bovine aortic endothelial cells, rabbit smooth muscle cells), exhibiting different cytoskeletal organizations and derived from several species, with doses of cytochalasin D (CD, 2-6 microM for 20 h) sufficient to induce cytoskeletal rearrangement and altered cellular morphology results in an increase in the relative content and rate of synthesis of actin. These data extend our previous findings for HEp-2 cells to other cell types and provide further evidence for our hypothesis that the CD-induced cytoskeletal reorganization triggers stimulation of actin synthesis and the resulting increase in actin content.     

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.     

Heat shock gene expression and cytoskeletal alterations in mouse neuroblastoma cells

The cytoskeleton of neuroblastoma cells, clone Neuro 2A, is altered by two stress conditions: heat shock and arsenite treatment. Microtubules are reorganized, intermediate filaments are aggregated around the nucleus, and the number of stress fibers is reduced. Since both stress modalities induce similar cytoskeletal alterations, no thermic denaturation of one or more cytoskeletal components can be involved in this process. Heat shock proteins are induced both by heat and by arsenite. However, cells treated with arsenite synthesize hsp28 which is not detected in heat-treated cells. Synthesis of all hsps is prevented by addition of actinomycin D or cycloheximide. Under these conditions no alterations are observed in the organization of microtubules and intermediate filaments during heat or arsenite treatment. However, these drugs are not able to prevent the rapid loss of stress fibers. A re-formation of the cytoskeleton during the recovery period proceeds within 3 h and is also found to occur in the presence of a protein synthesis inhibitor. These data suggest that reorganization of microtubules and intermediate filaments during a stress treatment requires the synthesis of a new protein(s), probably hsp(s).     

Cytoskeletal disruption accelerates caspase-3 activation and alters the intracellular membrane reorganization in DNA damage-induced apoptosis

In actinomycin D (AD)-induced apoptosis, caspase-3 activation and DNA cleavage in human megakaryoblastic leukemia CMK-7 cells were greatly accelerated by tubulin and actin polymerization inhibitors [e.g., colcemid (CL) and cytochalasin D (CD), respectively], but the acceleration was not found with Taxol or phalloidin. A decrease in mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of procaspase-9 to its active form preceded the activation of caspase-3 and, moreover, all of these events began earlier and/or proceeded faster in cells treated with AD plus CL or CD than in cells treated with AD only. These results suggest that cytoskeletal disruption in the apoptotic cells promotes damage of the mitochondrial membrane, resulting in the enhanced release of cytochrome c necessary for the activation of caspase-9 that initiates the caspase cascade. On the other hand, apoptotic bodies were rapidly formed from cells treated with AD and CL, but were suppressed when treated with AD and CD. Intracellular membranes and the actin system were reorganized to surround the nuclear fragments in the AD- and CL-treated cells, but such a membrane system was not formed in the presence of CD, implying that the apoptotic bodies are formed via reorganization of intracellular membranes under regulation by actin polymerization. Thus, the cytoskeletal change in CMK-7 cells has a strong effect on the early biochemical process as well as on the later morphologic process in AD-induced apoptosis.     

Effect of cytochalasin D on DNA synthesis in cultured cells

The effect of cytochalasin D (CD), an agent specifically destroying actin cytoskeleton, on DNA replication of cultured mouse embryonic fibroblasts (MEF) and BALB/3T3 strain cells was studied. Incubation of normal cells with CD resulted in progressive inhibition of DNA synthesis: in the first 16-20 h the percentage of cells pulse-labelled with 3H-thymidine was similar to that in control cultures, on day 8 the percentage of labelled cells was 7-8 times lower than in the control. The transfer of cells into fresh medium upon 8-day incubation in the presence of CD resulted in the recovery of DNA synthesis. Similar curves of DNA synthesis inhibition in the presence of CD and of DNA synthesis recovery in fresh medium were observed both in mononuclear and binuclear cells. Thus, CD-induced reorganization of actin cytoskeleton can have an abrupt but reversible disturbing effect on normal cell cycle.     

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.     

Development of cytoskeletal connections between cells of preimplantation mouse embryos

Summary Observations by scanning electron microscopy of mouse cleaving embryos reveal the presence of long microvilli around cell contact regions that often bridge the gap between blastomeres. These microvilli correspond, in detergent-extracted morulae, to strings connecting the cortical cytoskeletons of adjoining cells. They appear about 4 h after compaction in synchronized cultures. Transmission electron microscopy, heavy meromyosin decoration and DNase I digestion show that cytoskeletal connections contain bundles of actin microfilaments. The establishment of cytoskeletal connections does not require immediate protein synthesis, as shown by incubation with cycloheximide. Diverse treatments that interfere with compaction were tested for the development of cytoskeletal connections: culture media with low Ca²⁺ and/or Mg²⁺, or EGTA, or -lactalbumin, do not prevent the establishment of connections, while colchicine delays their appearance and cytochalasin D suppresses it. The relation between cytoskeletal connections, compaction and blastulation is discussed.     

Regulation of Herpesvirus Macromolecular Synthesis I. Cascade Regulation of the Synthesis of Three Groups of Viral Proteins

Based on evidence that 50% of herpes simplex 1 DNA is transcribed in HEp-2 cells in the absence of protein synthesis we examined the order and rates of synthesis of viral polypeptides in infected cells after reversal of cycloheximide- or puromycin-mediated inhibition of protein synthesis. These experiments showed that viral polypeptides formed three sequentially synthesized, coordinately regulated groups designated alpha, beta, and gamma. Specifically: (i) The alpha group, containing one minor structural and several nonstructural polypeptides, was synthesized at highest rates from 3 to 4 h postinfection in untreated cells and at diminishing rates thereafter. The beta group, also containing minor structural and nonstructural polypeptides, was synthesized at highest rates from 5 to 7 h and at decreasing rates thereafter. The gamma group containing major structural polypeptides was synthesized at increasing rates until at least 12 h postinfection. (ii) The synthesis of alpha polypeptides did not require prior infected cell protein synthesis. In contrast, the synthesis of beta polypeptides required both prior alpha polypeptide synthesis as well as new RNA synthesis, since the addition of actinomycin D immediately after removal of cycloheximide precluded beta polypeptide synthesis. The function supplied by the alpha polypeptides was stable since interruption of protein synthesis after alpha polypeptide synthesis began and before beta polypeptides were made did not prevent the immediate synthesis of beta polypeptides once the drug was withdrawn. The requirement of gamma polypeptide synthesis for prior synthesis of beta polypeptides seemed to be similar to that of beta polypeptides for prior synthesis of the alpha group. (iii) The rates of synthesis of alpha polypeptides were highest immediately after removal of cycloheximide and declined thereafter concomitant with the initiation of beta polypeptide synthesis; this decline in alpha polypeptide synthesis was less rapid in the presence of actinomycin D which prevented the appearance of beta and gamma polypeptides. The decrease in rates of synthesis of beta polypeptides normally occurring after 7 h postinfection was also less rapid in the presence of actinomycin D than in its absence, whereas ongoing synthesis of gamma polypeptides at this time was rapidly reduced by actinomycin D. (iv) Inhibitors of DNA synthesis (cytosine arabinoside or hydroxyurea) did not prevent the synthesis of alpha, beta, or gamma polypeptides, but did reduce the amounts of gamma polypeptides made.     

Actin Depolymerization Affects Stress-Induced Translational Activity of Potato Tuber Tissue

Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.     

Effect of modulators of cytoskeletal function on desensitization and recovery of PGE2-responsive ovarian adenylate cyclase.

Exposure of cultured Graafian follicles to PGE2 for 20 h resulted in a loss of the cyclic AMP response to fresh hormone. This desensitization was prevented by addition to the medium of D2O (25--50%) or Li+ (0.6--6 mM), agents believed to stabilize microtubules, as well as by phalloidin (1.0--10 microM), believed to stabilize the polymerized state of actin, in a dose-dependent manner. The spontaneous recovery of responsiveness to PGE2 upon incubation of refractory follicles for 6 h in hormone-free medium was prevented by addition to the medium of cytochalasin B (CB; 3 microgram/ml) or of the actin-binding myosin subfragment HMM S-1 (80 microgram/ml) or of anti-actin serum; viz. by agents likely to interfere with microfilament function. D2O (50%) caused morphological damage to the inner layer of the membrana granulosa and severe depression of protein synthesis. The other drugs used (phalloidin, LiCl and cytochalasin B) had no such effects. Resensitization of refractory follicles was also prevented by cycloheximide (10 micrograms/ml) and by actinomycin D (10 micrograms/ml). It is speculated that the recovery process may involve the insertion of a newly synthesized protein, such as PG-receptor, into the membrane by a mechanism dependent on microfilament action. These findings provide suggestive evidence for the hypothesis that cytoskeletal elements associated with the cell membrane take part in the modulation of the adenylate cyclase response to hormones.     

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.     

Endothelial actin cytoskeleton remodeling during mechanostimulation with fluid shear stress

Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Using the complementary techniques of photoactivation of fluorescence and fluorescence recovery after photobleaching, we have characterized endothelial actin cytoskeleton dynamics during the alignment process in response to steady laminar fluid flow and have correlated these results to motility. Alignment requires 24 h of exposure to fluid flow, but the cells respond within minutes to flow and diminish their movement by 50%. Although movement slows, the actin filament turnover rate increases threefold and the percentage of total actin in the polymerized state decreases by 34%, accelerating actin filament remodeling in individual cells within a confluent endothelial monolayer subjected to flow to levels used by dispersed nonconfluent cells under static conditions for rapid movement. Temporally, the rapid decrease in filamentous actin shortly after flow stimulation is preceded by an increase in actin filament turnover, revealing that the earliest phase of the actin cytoskeletal response to shear stress is net cytoskeletal depolymerization. However, unlike static cells, in which cell motility correlates positively with the rate of filament turnover and negatively with the amount polymerized actin, the decoupling of enhanced motility from enhanced actin dynamics after shear stress stimulation supports the notion that actin remodeling under these conditions favors cytoskeletal remodeling for shape change over locomotion. Hours later, motility returned to pre-shear stress levels but actin remodeling remained highly dynamic in many cells after alignment, suggesting continual cell shape optimization. We conclude that shear stress initiates a cytoplasmic actin-remodeling response that is used for endothelial cell shape change instead of bulk cell translocation. atherosclerosis; cytoskeletal dynamics; endothelial cells; mechanotransduction     

Stimulus-Induced Selective Association of Actin-Associated Proteins (α-Actinin) and Protein Kinase C Isoforms with the Cytoskeleton of Human Neutrophils

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein α-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal α-actinin and actin. Increased association of PKCβI and βII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, α-actinin, and PKCβII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal α-actinin and PKCβII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 μM) completely blocked PMA-induced increases in cytoskeletal α-actinin but reduced cytoskeletal recruitment of PKCβII only by 16%. Higher concentrations of latrunculin A (4 μM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCβII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

Regulation of actin mRNA levels and translation responds to changes in cell configuration.

The role of cell configuration in regulating cell metabolism has been studied, using a system in which cell shape and surface contact can easily be manipulated. The suspension of anchorage-dependent mouse fibroblasts in Methocel results in a coordinate decrease of DNA, RNA, and protein synthesis. These processes are restored upon reattachment of cells to a solid surface. This recovery process has two or more components: a rapid recovery of protein synthesis requiring only surface contact, and a slower restoration of nuclear events which is dependent upon extensive cell spreading (A. Ben-Ze'ev, S.R. Farmer, and S. Penman, Cell 21:365-372, 1980). In the present study, we examined 3T3 cells while in suspension culture and after attachment to a tissue culture dish surface to study cell configuration-dependent expression of specific cytoskeleton protein genes. The 3T3 line of fibroblasts used here shows these responses much more dramatically compared with 3T6 cells previously studied. We demonstrate that whereas total protein synthesis was strongly inhibited upon suspension, actin synthesis was preferentially inhibited, decreasing from 12% of total protein synthesis in control cells to 6% in suspended cells. This occurred apparently at the level of translation of actin mRNA, since the amount of actin mRNA sequences in the cytoplasm was unchanged. Reattachment initiated the rapid recovery of overall protein synthesis which was accompanied by a dramatic, preferential increase in actin synthesis reaching peak values of 20 to 25% of total protein synthesis 4 to 6 h later, but then declining to control values by 24 h. Translation in vitro and hybridization of mRNA to a cloned actin cDNA probe revealed that the induction of actin synthesis was due to increased levels of translatable mRNA sequences in the cytoplasm. These results imply a close relationship among cell cytoarchitecture, expression of a specific cytoskeletal protein gene, and growth control. The expression of the actin gene appears to be regulated at both the level of translation (during suspension) and mRNA production (during recovery).