microM Cytochalasin D Research Articles

Insulin stimulation of rat ventricular K+ currents depends on the integrity of the cytoskeleton.

1. The effect of insulin on K+ currents was studied with enzymatically dispersed ventricular myocytes from insulin-deficient (type I) diabetic rats. Diabetic conditions were induced by a single intravenous injection of streptozotocin (100 mg kg-1) given 8-13 days before the experiments. Measurements of plasma glucose and insulin levels confirmed the diabetic status of the animals. 2. A Ca2+-independent transient outward K+ current, It, and a slowly inactivating, quasi-steady-state current, Iss, which are depressed in diabetic myocytes, could be restored by exposure to 1, 10 or 100 nM insulin. This was only observed after a delay of 5-6 h, although an insulin exposure of only 1 h was sufficient to initiate its stimulatory action on It and Iss. The stimulatory effect of insulin on these K+ currents was prevented by 2 microM cycloheximide, which in itself had no direct effect on these currents. 3. Disruption of the actin microfilament network with 1 microM cytochalasin D (CD) also prevented the stimulatory effect of 100 nM insulin on both It and Iss. Since CD was added 1 h after insulin, inhibitory effects on insulin signalling were ruled out. Adding CD (1 microM) 5-9 h after insulin, when currents were already augmented, had no effect (up to 50 min exposure). Incubating control cells for 6-10 h with 1 microM CD had no effect on any of the currents measured. 4. Stabilization of the actin network by pre-exposure to 2.5 microM phalloidin restored the stimulatory effect of insulin, in the continued presence of CD, ruling out any effects of CD on components other than the cytoskeleton. 5. The stimulatory effect of insulin was also prevented by incubating cells with insulin in the presence of the microtubule-disrupting agent colchicine (5 microM). 6. These results suggest that the insulin-mediated augmentation of K+ currents in diabetic myocytes requires protein synthesis, possibly of K+ channels, as well as an intact cytoskeleton. The possibility that newly formed channels translocate to the plasma membrane in a process dependent on different elements of the cytoskeleton is discussed.

Chromosome movement during meiotic prophase in crane-fly spermatocytes: IV. Actin and the effects of cytochalasin D

Cytochalasin D (CD) was applied to crane-fly spermatocytes at late diakinesis with the aim of perturbing actin structure and actin function, thereby testing the hypothesis that intranuclear chromosome movement during late diakinesis is actin-based. Isolated tests were incubated in a range of CD concentrations (2-100 microM) for 1 or 2 h. None of those treatments resulted in cessation of prophase movements in living cells. An immediate effect of 10-100 microM CD at late diakinesis was the formation of highly refractile, actin-containing cables within the nonchromosomal nucleoplasm. No such cables were observed in vehicle-treated control cells. CD treatments caused autosomal bivalents in unusually large numbers of spermatocytes to become aggregated into densely-packed clusters; for example, with 40 microM CD about 80% of late diakinesis spermatocytes had clustered autosomes, vs. about 25% clustering in untreated cells. We conclude from these data that the mechanism of chromosome positioning at the nuclear envelope is CD-sensitive. Rhodamine-conjugates of phalloidin and DNase I were used to assess the status of actin in untreated cells as well as the effect of CD on actin distribution. Differences in nucleoplasmic staining with phalloidin and DNase I conjugates suggest that nucleoplasm at late diakinesis contains actin in a nonfilamentous form.

Effect of cytochalasin D on the mechanical properties and morphology of passive human neutrophils.

The actin-rich cortex plays a major role in neutrophil chemotaxis and phagocytosis. In passive neutrophils, 30-50% of the actin molecules are in the F (filamentous) form, and it is the shifting of equilibrium with its monomeric G (globular) form that controls cell motility and phagocytosis. Cytochalasins have been shown to inhibit cell phagocytosis and ruffling. In purified actin, cytochalasins have been shown to decrease the amount of F-actin by capping the fast-growth end of actin filaments. Recent studies with intact cells, however, reveal that the most potent cytochalasin, cytochalasin D (CD), actually increases F-actin content suggesting that CD disrupts the actin network so as to increase the number of actin-filament ends for further actin polymerization. In this paper, we report the effects of CD on the passive mechanical behavior and morphology of human neutrophils with 1, 2, 10, and 20 microM CD. At 1 and 2 microM CD, the cells remain spherical. However, in the presence of 10 and 20 microM CD, cells are severely deformed and "blebby" as shown by light and scanning electron microscopy. After 1 and 2 microM CD treatment, the cells show a decrease of 43 and 66%, respectively, in cortical tension when measured by static micropipet aspiration experiments. Similarly, the cytoplasmic viscosities of 1 and 2 microM CD-treated cells are decreased, but only by 17 and 24%, respectively. A proportionally greater effect on the cortical tension suggests that CD acts mainly on the actin-rich cortex by disrupting the filament network.

Effect of cytochalasin D on the actin cytoskeleton of the toad bladder epithelial cell.

Cytochalasins are widely used to determine the role of actin in cellular processes. Their actions include capping of the barbed end of actin filaments as well as dimer formation, nucleation, and polymerization. We determined the effect of cytochalasin D (CD) on F-actin in the toad urinary bladder, an epithelium in which vasopressin depolymerizes F-actin. At a low concentration (0.25 microM), CD depolymerized F-actin in the unstimulated cell; at higher concentrations, there was a progressive reduction of depolymerization until actual polymerization was seen. Vasopressin plus CD produced no greater depolymerization than vasopressin alone, suggesting that CD and vasopressin act to a large extent on the same pool of F-actin. CD plus vasopressin also enhanced the fusion rate of aggrephores compared with vasopressin alone, indicating that intact actin filaments retard aggrephore fusion. Despite the increase in aggrephore fusion, water flow was not enhanced by CD, confirming previous reports that intact actin filaments are required for water channel emergence or stabilization in the apical membrane. Vasopressin plus 1 microM CD produced a striking increase in microvillar length, direct evidence of the polymerizing action of CD in the cell.

Effects of cytochalasin, phalloidin and pH on the elongation of actin filaments

We used electron microscopy to measure the effects of cytochalasins, phalloidin, and pH on the rates of elongation at the barbed and pointed ends of actin filaments. In the case of the cytochalasins, we compared the effects on ATP- and ADP-actin monomers. Micromolar concentrations of either cytochalasin B (CB) or cytochalasin D (CD) inhibit elongation at both ends of the filament, about 95% at the barbed end and 50% at the pointed end, so that the two ends contribute about equally to the rate of growth. Half-maximal inhibition of elongation at the barbed end is at 0.1 microM CB and 0.02 microM CD for ATP-actin and at 0.1 microM CD for ADP-actin. At the pointed end, CD inhibits elongation by ATP-actin and ADP-actin about equally. At high (2 microM) concentrations, the cytochalasins reduce the association and dissociation rate constants in parallel for both ADP- and ATP-actin, so their effects on the critical concentrations are minimal. These observations confirm and extend those of Bonder and Mooseker [Bonder, E. M., & Mooseker, M. S. (1986) J. Cell Biol. 102, 282-288]. The dependence of the elongation rate on the concentration of both cytochalasin and actin can be explained quantitatively by a mechanism that includes the effects of cytochalasin binding to actin monomers [Godette, D. W., & Frieden, C. (1986) J. Biol. Chem. 261, 5974-5980] and a partial cap of the barbed end of the filament by the complex of ADP-actin and cytochalasin.(ABSTRACT TRUNCATED AT 250 WORDS)

P52 induction by cytochalasin D in rat kidney fibroblasts: Homologies between p52 and plasminogen activator inhibitor type‐1

Normal rat kidney (NRK) fibroblasts respond to the cell shape-modulating chemical agent cytochalasin D (CD) with augmented synthesis of the 52-kDa substrate-associated protein p52. p52 is a complex glycoprotein, existing as 12 different isoforms, which include a 43-kDa "core" protein (p43), four 50-kDa species (p50-0,1,2,3), and at least seven distinct pI variants of the mature 52-kDa protein. A threshold of 2-4 microM CD was found to be necessary to augment p52 deposition into both the secreted protein- and saponin-resistant cytomatrix (SAP) fractions of NRK cells. This concentration of CD was also necessary to initiate significant cell rounding. Augmented p52 production in CD-treated NRK (NRK/CD) cells provided a means to assess the identity of this protein. p52 was found to be identical to rat plasminogen activator inhibitor type-1 (rPAI-1) and to PAI-1-like proteins of other species by comparative immunoprecipitation, 2-D electrophoretic profile, V8 protease digest mapping, and subcellular fractionation criteria. Quantitation of rPAI-1 cytoplasmic mRNA abundance, using the rPAI-1 cDNA probe pSS1-3, revealed an induction of rPAI-1 mRNA in NRK/CD cells which paralleled the increased protein production. CD-augmented p52(rPAI-1) synthesis and SAP deposition was blocked by actinomycin D, implicating a need for RNA synthesis during the period of CD exposure to effect induction. Augmentation of p52 expression in NRK/CD fibroblasts, thus, appears to involve both cell shape-associated metabolic processes and concomitant RNA synthesis.

Cytochalasin D-mediated hyperinduction of the substrate-associated 52-kilodalton protein p52 in rat kidney fibroblasts.

Regulation of certain differentiated and housekeeping functions in cultured mammalian cells is significantly influenced by cell shape. The shape-modulating agent cytochalasin D (CD) was used, therefore, to elucidate potential cytoarchitectural influences affecting synthesis of a major 52 kDa secreted/substrate-associated protein (p52) of normal rat kidney (NRK) fibroblasts. Biosynthetic labeling experiments indicated that treatment of NRK cells with CD increased, by 10-18-fold, the medium content of an Mr 52,000 protein. Two-dimensional gel electrophoresis and peptide fragment mapping confirmed that the 52 kDa protein produced in abundance as a consequence of CD treatment was identical to p52 constitutively expressed by NRK cells. A lower mw protein (p50; Mr 50,000) was also resolved which, based on pl microheterogeneity, protease fragmentation profile, and sensitivity to tunicamycin, could be identified as a less-glycosylated form of p52. p50 and p52 were both detected in the matrix and medium compartments of NRK and NRK/CD cells. The matrix p52 content of CD-induced and uninduced cells, however, was significantly greater (by 200-500-fold) than the corresponding medium levels. This differential compartmentalization, the time course of p52 accumulation in the matrix of NRK/CD cells compared to its appearance in the medium, and the kinetics of p52 pulse-chase from the matrix collectively indicated that the matrix is the initial site of p52 deposition. Low levels of CD (1 microM) produced extensive disruptions of cellular microfilaments but did not result in an overall cell shape change nor a hyperinduction of p52. Morphologic rounding (seen in 10-100 microM CD) coincided with augmented p52 production. Transition from a flat to a round phenotype in NRK cells, or at least the generation of sufficient microfilament fragmentation to compromise cell-substrate adhesivity, appears to be an essential aspect of CD-mediated p52 hyperinduction.

Effects of cytochalasin D on the integrity of the Sertoli cell (blood-testis) barrier.

Ectoplasmic specializations (ES) containing packed actin microfilaments are associated with the numerous parallel rows of occluding junctions which form the Sertoli cell (blood-testis) barrier. To determine if ES regulate the structure of the occluding junctions and/or barrier permeability, we experimentally disrupted ES microfilaments in vivo with intratesticularly injected cytochalasin D (CD). Electron microscopic observations of seminiferous tubules from CD-treated (150-500 microM CD; 0.5-12 hr) animals indicated that ES was absent from regions where the Sertoli cell barrier is located. Seminiferous epithelial sheets from uninjected or vehicle-injected animals (1 DMSO: 1 saline) stained with NBD-phallacidin demonstrated the presence of patterned ES actin surrounding the basolateral regions of adjacent Sertoli cells. After exposure to CD, epithelial sheets exhibited increasingly patchy fluorescence indicating progressive F-actin disruption. Freeze-fracture replicas of CD-injected testes revealed numerous focal alterations in the region of occluding junctions which included disorganization of the parallel arrangement of junctional rows, the presence of free-ending rows, clustering of intramembranous particles (IMPs) between rows, reduction in the number of rows, and loss of IMPs on both the P-face and E-face. Tracer experiments, following CD exposure, were conducted to test the integrity of occluding junctions: lanthanum hydroxide, dextrose, or filipin was added, in separate experiments, to the fixative during perfusion-fixation. In another study, serum containing an antibody against adluminal germ cells was injected intratesticularly, and frozen sections were processed for immunofluorescence study. A final study consisted of simultaneous intratesticular infusions of CD and radiolabelled inulin with subsequent intraluminal and peritubular fluid sampling. In animals which were injected with CD, lanthanum was found to enter the adluminal compartment; fixative made hypertonic by addition of dextrose caused germ cells within the adluminal compartment to shrink and produce exaggerated intercellular spaces; filipin-cholesterol perturbations were present between some Sertoli cell junctional rows and on spermatid plasma membranes; and IgG was detected within the adluminal compartment of many seminiferous tubules. None of these adluminal manifestations was noted in control animals or those which received vehicle. Quantitatively, in the in vivo micropuncture experiments, significantly more radiolabelled inulin entered the lumen of seminiferous tubules from CD-treated animals than from those exposed to vehicle.(ABSTRACT TRUNCATED AT 400 WORDS)

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.