Effect of cytochalasins on F-actin and morphology of Ehrlich ascites tumor cells

Cytochalasins have been used extensively to probe the role of F-actin in different aspects of cellular function. Most of the data obtained are interpreted on the basis of the well-established depolymerizing effects of cytochalasins on F-actin preparations in vitro. However, some evidence indicates that, in intact cells, different cytochalasins can have varying effects on cell morphology and F-actin content and organization. To examine this problem in more detail, we analyzed the effects of cytochalasins on the cell morphology of and F-actin content and organization in Ehrlich ascites tumor (EAT) cells. After a 3-min exposure to 0.5 microM cytochalasin D, B, or E, F-actin content was equally reduced in all cases and this correlated with a reduction in the amount of cortical F-actin associated with the EAT cell membrane. However, only with CE was cell morphology markedly altered, with the appearance of numerous blebs. At 10 microM, blebbing was present in all conditions and the organization of cortical F-actin was disrupted. F-actin content, however, was not further reduced by this higher concentration and in CD it was identical to control levels. Exposure of EAT cells to similar concentrations of cheatoglobosin C, an analog of the cytochalasins that has little to no affinity for F-actin, resulted in a loss of F-actin content, a reduction in F-actin fluorescence, but no change in cell morphology, including a complete lack of bleb formation. Myosin II immunoreactivity, concentrated in the cortical cytoplasm colocalized with F-actin and in an area associated with the Golgi, was reduced by the high-dose cytochalasin. These results demonstrate that caution must be exercised in the use of cytochalasins to probe the role of F-actin in cellular function and that several parameters must be analyzed to obtain an accurate assessment of the effect of cytochalasin on the actin filament system.     

Cytochalasins induce actin polymerization in human leukocytes.

We studied the effect of cytochalasins (B, D, and E) on the F-actin content in human neutrophils and lymphocytes using NBD-phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration- and time-dependent increase in the F-actin content in both cell types. The order of potency was cytochalasin D greater than E greater than B. The increase in F-actin content was accompanied by a decrease in the G-actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin-binding or sequestering proteins. 2-deoxyglucose (20 mM) caused a decrease in the basal F-actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.     

Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro

To compare the effects of cytochalasins on the cellular level with those on the molecular level, 24 cytochalasins, 20 natural compounds and 4 derivatives, were used. The following effects were tested for each of 24 cytochalasins; (a) four high dose (2-20 muM) effects on the cellular level: rounding up of fibroblastic cells, contraction of actin cables, formation of hairy filaments containing actin, and inhibition of lymphocyte capping; (b) a low dose (0.2-2 muM) effect: inhibition of membrane ruffling; and (c) two in vitro effects: an inhibition of actin filament elongation (the high affinity effect [low dose effect] in vitro) and an effect on viscosity of actin filaments(the low affinity effect [high dose effect] in vitro). These results indicated that there are almost the same hierarchic orders of relative effectiveness of different cytochalasins between low and high dose effects and between cellular and molecular effects. From the data obtained with the 24 cytochalasins, we have calculated correlation coefficients of 0.87 and 0.79 between an effect in vivo, inhibition of capping, and an effect in vitro, inhibition of actin filament elongation, as well as between inhibition of capping and another effect in vitro, effect on viscosity of actin filaments, respectively. Furthermore, a correlation coefficient between the high affinity effect and the low affinity effect determined in vitro was calculated to be 0.90 from the data obtained in this study. The strong positive correlation among low and high dose effects in vivo and those in vitro suggests that most of the effects caused by a cytochalasin, irrespective of doses or affected phenomena, might be attributed to the interaction between the drug and the common target protein, actin. In the course of the immunofluorescence microscope study on cytochalasin-treated cells using actin antibody, we have found that aspochalasin D, a 10-isopropylcytochalasin, strongly induced the formation of rodlets containing actin in the cytoplasm of the treated fibroblasts. In contrast, the other cytochalasins, including cytochalasin B, cytochalasin C, cytochalasin D, and cytochalasin H, were found to induce the formation of nuclear rodlets. Both cytoplasmic and nuclear rodlets found in the cytochalasin-treated cells were similar in ultrastructures to those induced by 5 to 10 percent (vol/vol) dimethyl sulfoxide in the same type of cells.     

Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation

Polylysine was found to induce polymerization of muscle actin in a low ionic strength buffer containing 0.4 mM MgCl2. The rate of induced polymerization was dependent on the amount and on the molecular size of the polylysine added. A similar effect was obtained by adding actin nuclei (containing about 2-4 actin subunits) cross-linked by p-N,N'- phenylenebismaleimide to G-actin under the same conditions, suggesting that the effect of polylysine is due to promotion of the formation of actin nuclei. Polymerization induced by polylysine and by cross-linked actin nuclei was inhibited by low concentrations (10(-8)-10(-6)M) of cytochalasins. Binding experiments showed that actin filaments, but not actin monomers, contained high-affinity binding sites for [3H]cytochalasin B (one site per 600 actin monomers). The relative affinity of several cytochalasins for these sites (determined by competitive displacement of [3H]dihydrocytochalasin B) was: cytochalasin D greater than cytochalasin E approximately equal to dihydrocytochalasin B. The results of this study suggest that cytochalasins inhibit nuclei-induced actin polymerization by binding to highly specific sites at the point of monomer addition, i.e., the elongation site, in actin nuclei and filaments.     

Spectrin-4.1-actin complex of the human erythrocyte: Molecular basis of its ability to bind cytochalasins with high-affinity and to accelerate actin polymerization in vitro

The spectrin-4.1-actin complex isolated from the cytoskeleton of human erythrocyte [3] was found to be similar to muscle F-actin in several aspects: Both the complex and F-actin nucleate cytochalasin-sensitive actin polymerization; both bind dihydrocytochalasin B with similar binding constants; both can be depolymerized by DNase I with loss of cytochalasin binding activity. From these results, we conclude that the actin in the complex is in an oligomeric form. However, the presence of spectrin and band 4.1 in the complex not only stabilized the actin in the complex as evidenced by its resistance to depolymerization in low-ionic-strength conditions and to DNase I as compared with F-actin, but also altered the characteristics of the binding site(s) for cytochalasins believed to be located at the “barbed” (polymerizing) end of the oligomeric actin.     

The cytoskeleton and cell volume regulation

Although the precise mechanisms have yet to be elucidated, early events in osmotic signal transduction may involve the clustering of cell surface receptors, initiating downstream signaling events such as assembly of focal adhesion complexes, and activation of, e.g. Rho family GTPases, phospholipases, lipid kinases, and tyrosine- and serine/threonine protein kinases. In the present paper, we briefly review recent evidence regarding the possible relation between such signaling events, the F-actin cytoskeleton, and volume-regulatory membrane transporters, focusing primarily on our own work in Ehrlich ascites tumer cells (EATC). In EATC, cell shrinkage is associated with an increase, and cell swelling with a decrease in F-actin content, respectively. The role of the F-actin cytoskeleton in cell volume regulation in various cell types has largely been investigated using cytochalasins to disrupt F-actin and highly varying effects have been reported. Findings in EATC show that the effect of cytochalasin treatment cannot always be assumed to be F-actin depolymerization, and that, moreover, there is no well-defined correlation between effects of cytochalasins on F-actin content and their effects on F-actin organization and cell morphology. At a concentration verified to depolymerize F-actin, cytochalasin B (CB), but not cytochalasin D (CD), inhibited the regulatory volume decrease (RVD) and regulatory volume increase (RVI) processes in EATC. This suggests that the effect of CB is related to an effect other than F-actin depolymerization, possibly its F-actin severing activity.     

Regulatory role of microfilaments in the induction of T4 cell proliferation and interleukin 2 production

The role of microfilaments in human T4 cell proliferation and lymphokine production triggered via various pathways of activation was examined by investigating the effects of cytochalasins on these responses. The data demonstrate that the effects of cytochalasins vary depending on the nature of the stimulus and on the concentration of the cytochalasin. Concentrations of cytochalasin that would be expected to bind both the low and high affinity binding sites (5-20 microM), that represent cytosolic and surface actin filaments, respectively inhibited T4 cell proliferation regardless of the stimulus. T4 cell proliferation stimulated by antigen-bearing APC or anti-CD3 was inhibited much more markedly than responses stimulated by ionomycin and PMA. In contrast, concentrations of cytochalasin expected to bind only high affinity binding sites (0.125-1 microM), represented by surface actin filaments, enhanced T4 cell proliferation and interleukin 2 production stimulated by mAb to CD2, CD3, or class I major histocompatibility complex (MHC) molecules, but not those induced by mAb to the T cell receptor, paraformaldehyde fixed, or viable antigen-bearing APC, allogeneic APC, or ionomycin and PMA. The enhancing effect of cytochalasins on responses stimulated by cross-linking class I MHC molecules was studied in detail. Enhancement of T4 cell proliferation induced in this manner required that cytochalasin B was present between 4 and 18 hr of culture, but not before or after. The data demonstrate that T cell microfilaments play a number of roles in determining the magnitude of T cell responses induced by engaging specific cell surface receptors and imply that different components of the microfilament system exert opposing intrinsic regulatory effects on T cell function.     

The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.     

Latrunculins--novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D

The latrunculins are architecturally novel marine compounds isolated from the Red Sea sponge Latrunculia magnifica. In vivo, they alter cell shape, disrupt microfilament organization, and inhibit the microfilament-mediated processes of fertilization and early development. In vitro, latrunculin A was recently found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 molar complex with G-actin. These in vitro effects as well as previous indications that the latrunculins are more potent than the cytochalasins suggest differences in the in vivo mode of action of the two classes of drugs. To elucidate these differences we have compared the short- and long-term effects of latrunculins on cell shape and actin organization to those of cytochalasin D. Exposure of hamster fibroblast NIL8 cells for 1-3 hr to latrunculin A, latrunculin B, and cytochalasin D causes concentration-dependent changes in cell shape and actin organization. However, the latrunculin-induced changes were strikingly different from those induced by cytochalasin D. Furthermore, while initial effects were manifest with both latrunculin A and cytochalasin D already at concentrations of about 0.03 microgram/ml, latrunculin A caused complete rounding up of all cells at 0.2 microgram/ml, whereas with cytochalasin D maximum contraction was reached at concentrations 10-20 times higher. The short-term effects of latrunculin B were similar to those of latrunculin A although latrunculin B was slightly less potent. All three drugs inhibited cytokinesis in synchronized cells, but their long-term effects were markedly different. NIL8 cells treated with latrunculin A maintained their altered state for extended periods. In contrast, the effects of cytochalasin D progressed with time in culture, and the latrunculin B-induced changes were transient in the continued presence of the drug. These transient effects were found to be due to a gradual inactivation of latrunculin B by serum and were used to compare recovery patterns of cell shape and actin organization in two different cell lines. This comparison showed that the transient effects of latrunculin B were fully reversible for the NIL8 cells and not for the mouse neuroblastoma N1E-115 cells.     

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.     

The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils

Formyl-met-leu-phe (fMLP) induces actin assembly in neutrophils; the resultant increase in F-actin content correlates with an increase in the rate of cellular locomotion at fMLP concentrations less than or equal to 10(-8) M (Howard, T.H., and W.H. Meyer, 1984, J. Cell Biol., 98:1265-1271). We studied the time course of change in F-actin content, F-actin distribution, and cell shape after fMLP stimulation. F-actin content was quantified by fluorescence activated cell sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells (Howard, T.H., 1982, J. Cell Biol., 95(2, Pt. 2:327a). F-actin distribution and cell shape were determined by analysis of fluorescence photomicrographs of nitrobenzoxadiazole-phallacidin-stained cells. After fMLP stimulation at 25 degrees C, there is a rapid actin polymerization that is maximal (up to 2.0 times the control level) at 45 s; subsequently, the F-actin depolymerizes to an intermediate F-actin content 5-10 min after stimulation. The depolymerization of F-actin reflects a true decrease in F-actin content since the quantity of probe extractable from cells also decreases between 45 s and 10 min. The rate of actin polymerization (3.8 +/- 0.3-4.4 +/- 0.6% increase in F-actin/s) is the same for 10(-10) - 10(-6) M fMLP and the polymerization is inhibited by cytochalasin D. The initial rate of F-actin depolymerization (6.0 +/- 1.0-30 +/- 5% decrease in F-actin/min) is inversely proportional to fMLP dose. The F-actin content of stimulated cells at 45 s and 10 min is greater than control levels and varies directly with fMLP dose. F-actin distribution and cell shape also vary as a function of time after stimulation. 45 s after stimulation the cells are rounded and F-actin is diffusely distributed; 10 min after stimulation the cell is polarized and F-actin is focally distributed. These results indicate that actin polymerization and depolymerization follow fMLP stimulation in sequence, the rate of depolymerization and the maximum and steady state F-actin content but not the rate of polymerization are fMLP dose dependent, and concurrent with F-actin depolymerization, F-actin is redistributed and the cell changes shape.     

Chemoattractant-stimulated polymorphonuclear leukocytes contain two populations of actin filaments that differ in their spatial distributions and relative stabilities

Chemoattractants stimulate actin polymerization in lamellipodia of polymorphonuclear leukocytes. We find that removal of chemoattractant results in rapid (within 10 s at 37 degrees C) and selective depolymerization of the F-actin located in lamellipodia. Addition of 10 microM cytochalasin B, in the presence of chemoattractant, also resulted in rapid and selective depolymerization of lamellar F-actin. The elevated F-actin level induced by chemoattractant rapidly returns to the level present in unstimulated cells after (a) a 10-fold decrease in chemoattractant concentration; (b) the addition of 10 microM cytochalasin B; or (c) cooling to 4 degrees C. The F-actin levels of unstimulated cells are only slightly affected by these treatments. Based on the similar effects of cytochalasin addition and chemoattractant dilution, it is likely that both treatments result in actin depolymerization from the pointed ends of filaments. Based on our results we propose that chemoattractant-stimulated polymorphonuclear leukocytes contain two distinct populations of actin filaments. The actin filaments within the lamellipodia are highly labile and in the continued presence of chemoattractant these filaments are rapidly turning over, continually polymerizing at their plus (barbed) ends, and depolymerizing at their minus ends. In contrast, the cortical F-actin filaments of both stimulated and unstimulated cells are differentially stable.     

Volume regulation in leukocytes: Requirement for an intact cytoskeleton

Animal cells regulate their volume by controlling the flux of ions across their plasma membrane. Recent evidence suggests that ion channels and pumps are physically associated with, and may be regulated by components of the cytoskeleton. To elucidate the role of elements of the cytoskeleton in volume regulation, we studied the effects of cytoskeletal disrupting agents on regulatory volume decrease (RVD) in three different leukocyte types: Jurkat lymphoma cells, HL-60 cells, and human peripheral blood neutrophils. Cell volume was measured in two ways: (i) electronically with a Coulter counter and (ii) by forward light scattering in a flow cytometer. Exposure of all leukocyte types to hypotonic medium (200 mOsm) resulted in an immediate increase in cell volume followed by a regulatory decrease to baseline by 20 min. In the presence of the microtubule disrupting agents, colchicine and nocodazole, RVD was totally inhibited which corresponded to loss of microtubules as determined by immunofluorescence. Similarly, RVD was inhibited in Jurkat cells incubated with the actin binding agents, cytochalasin B (CB) or D (CD). In contrast, in HL-60 cells and human neutrophils, RVD was unaffected by treatment with either CB or CD. While cytochalasins are generally thought of as microfilament disrupting agents, their primary action is to prevent F-actin polymerization. The extent of ensuing microfilament disruption depends in part on the rate of filament turnover. In an attempt to understand the differential effects of the cytochalasins on RVD, the F-actin content of the different cells was determined by NBD-phallacidin staining and flow cytometry. Pretreatment with CB or CD resulted in profound actin disassembly in Jurkat cells (relative fluorescence index RFI: 1.0 control vs. 0.21 ± 0.01 for CB and 0.48 ± 0.02 for CD). However, the cytochalasins did not induce net disassembly in either HL-60 cells or human neutrophils. To study the effects of an increase in F-actin on volume regulation, neutrophils were treated with the chemoattractant f-Met-Leu-Phe or with an antibody (Ab) to β2 integrins followed by a cross-linking secondary Ab. Despite an increase in F-actin in both circumstances, RVD remained intact. Taken together, these results suggest that both microtubules and microfilaments are important in volume regulation. © 1995 Wiley-Liss, Inc.     

Regulation of actin dynamics is critical for endothelial barrier functions

We tested the hypothesis that the equilibrium between F- and G-actin in endothelial cells modulates the integrity of the actin cytoskeleton and is important for the maintenance of endothelial barrier functions in vivo and in vitro. We used the actin-depolymerizing agent cytochalasin D and jasplakinolide, an actin filament (F-actin) stabilizing and promoting substance, to modulate the actin cytoskeleton. Low doses of jasplakinolide (0.1 microM), which we have previously shown to reduce the permeability-increasing effect of cytochalasin D, had no influence on resting permeability of single-perfused mesenteric microvessels in vivo as well as on monolayer integrity. The F-actin content of cultured endothelial cells remained unchanged. In contrast, higher doses (10 microM) of jasplakinolide increased permeability (hydraulic conductivity) to the same extent as cytochalasin D and induced formation of intercellular gaps in cultured myocardial endothelial (MyEnd) cell monolayers. This was accompanied by a 34% increase of F-actin and pronounced disorganization of the actin cytoskeleton in MyEnd cells. Furthermore, we tested whether an increase of cAMP by forskolin and rolipram would prevent the cytochalasin D-induced barrier breakdown. Conditions that increase intracellular cAMP failed to block the cytochalasin D-induced permeability increase in vivo and the reduction of vascular endothelial cadherin-mediated adhesion in vitro. Taken together, these data support the hypothesis that the state of polymerization of the actin cytoskeleton is critical for maintenance of endothelial barrier functions and that both depolymerization by cytochalasin D and hyperpolymerization of actin by jasplakinolide resulted in an increase of microvessel permeability in vivo. However, cAMP, which is known to support endothelial barrier functions, seems to work by mechanisms other than stabilizing F-actin.     

Effects of chaetoglobosin J on the G-F transformation of actin

It was shown that substoichiometric concentrations of chaetoglobosin J, one of the fungal metabolites belonging to cytochalasins, inhibited the elongation at the barbed end of an actin filament. Stoichiometric concentrations of chaetoglobosin J decreased both the rate and the extent of actin polymerization in the presence of 75 mM KCl, 0.2 mM ATP and 10 mM Tris-HCl buffer at pH 8.0 and 25 degrees C. In contrast, stoichiometric concentrations of cytochalasin D accelerated actin polymerization. Chaetoglobosin J slowly depolymerized F-actin to G-actin until an equilibrium was reached. Analyses by a number of different methods showed the increase of monomer concentration at equilibrium to depend on chaetoglobosin J concentrations. F-actin under the influence of stoichiometric concentrations of chaetoglobosin J only slightly activated the Mg2+-enhanced ATPase activity of myosin at low ionic strength. It is suggested that when the structure of the chaetoglobosin-affected actin filaments is modified, the equilibrium is shifted to the monomer side, and the interaction with myosin is weakened.     

Cryptotanshinone inhibits macrophage migration by impeding F-actin polymerization and filopodia extension

We evaluated the anti-inflammatory effects of cryptotanshinone and tanshinone IIA, two major tanshinones isolated from Salvia miltiorrhiza, on chemoattractant-induced cell migration in RAW264.7 macrophages. Results showed that cryptotanshinone inhibited cell migration toward complement 5a (C5a) and macrophage inflammatory protein-1alpha (MIP-1alpha) in a concentration-dependent manner. In contrast, tanshinone IIA displayed less or even no effect on cell migration evoked by these chemoattractants. Both C5a- and MIP-1alpha-induced migration were clearly inhibited by cytochalasin B (an inhibitor of actin polymerization), but not by colchicine (an inhibitor of microtubule polymerization). Fluorescence staining demonstrated that cryptotanshinone as well as cytochalasin B, effectively reversed cell polarization and filopodia extension induced by both chemoattractants. Furthermore, C5a-evoked increase in F-actin fluorescence intensity was significantly suppressed by cryptotanshinone. Based on these observations, we suggest that cryptotanshinone exerts anti-migrating activity possibly by impeding F-actin polymerization and filopodia formation.     

Effect of cytochalasin B on formation and properties of muscle F-actin.

Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(alpha-aminoethyl ether)-N,N'-tetraacetic acid and reduced by MgCl2 at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B.     

Specific interaction of cytochalasins with muscle and platelet actin filaments in vitro

The cytochalasins (CE, CD, CB and H₂CB) inhibit numerous cellular processes which require the interaction of actin with other structural and contractile proteins. In this report we describe the effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin. The cytochalasins decreased the viscosity of F-actin solutions. The effect of H₂CB, CB and CD on F-actin viscosity was maximal at concentrations of 20–50μM and did not increase with time. In contrast, CE caused a progressive decrease in the viscosity of F-actin solutions which was dependent upon the concentration of CE and the duration of incubation of the CE-actin mixture. After two hours of incubation of drug-actin mixtures, the relative effectiveness of the cytochalasins in reducing the viscosity of F-actin was CE > CD > CB = H₂CB. The effects of CD and CE were paralleled by morphologic changes in negatively stained actin filaments. The effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin were the same whether the drugs were added before or after the polymerization of the protein. These studies show that the interaction of the cytochalasins with actin is highly specific. Because the relative potencies of these drugs for affecting motile processes and the relative affinities of the drugs for binding sites within a variety of cells are CE > CD > CB = H₂CB, the effects of cytochalasins on actin described here may contribute to some of the biological effects of the drugs on motile processes.     

Cytochalasins distinguish by their action resting human T lymphocytes from activated T-cell blasts

In the majority of resting human peripheral T lymphocytes obtained from separate individuals cytochalasin B (CB) and D (CD) cause a disappearance of microvilli and induce a rapid formation of prominent sac and bleb-like projections with a length of 1–10 μm randomly distributed over the cell surface. During mitogen stimulation the cells lose the tendency to develop such projections when subsequently exposed to CB and CD. By contrast, in activated T lymphocytes the cytochalasins provoke an asymmetric localization of microvilli including cell surface antigens and actin to a prominent protuberance often separated from the cell body by a constriction. This protuberance is distinct from conventional spontaneous uropods formed by conA-stimulated lymphocytes in relation to contact with other cells and with non-cellular surfaces. The cytochalasins therefore in their action distinguish resting small lymphocytes from activated T-cell blasts.     

Effects of Multidrug Resistance-Related ATP-Binding-Cassette Transporter Proteins on the Cytoskeletal Activity of Cytochalasins

Cytochalasins are microfilament-active mould metabolites, widely utilized to study the involvement of the actin cytoskeleton in cellular processes as well as in genotoxicity and cell kinetic research. In this study we have investigated whether multidrug-resistance phenotypes, caused by overexpression of the ATP-binding-cassette transporter proteins P-glycoprotein (P-gp) or multidrug-resistance-associated protein (MRP), influence the microfilament-depolymerizing effect of cytochalasins. Using four well-characterized multidrug-resistance cell models, we have shown that both the microfilament-disrupting (phalloidine staining) and the cytotoxic (MTT-assay) activity of cytochalasins are reduced in parallel with increased P-gp expression and restorable by P-gp-modulating agents. This also applied to the cytochalasin D-mediated induction of polykaryons (microscopic evaluation) which arise as a consequence of impaired cytokinesis but unaffected karyokinesis. The reduced cellular activity of cytochalasins in P-gp-positive cell lines was correlated with decreased intracellular accumulation ([³H]cytochalasin B accumulation) which was also restorable by P-gp modulators. Moreover, the dose-dependent inhibition of P-gp photoaffinity labeling ([³H]azidopine) suggested cytochalasins as P-gp-binding agents. In contrast, MRP overexpression had no effect on either cytochalasin microfilament activity or cytotoxicity. In conclusion, data indicate that the microfilament-destructive effects of cytochalasins are impaired due to a reduction of the intracellular cytochalasin accumulation by P-gp but not by MRP. Results are discussed with regard to P-gp as a resistance factor when cytochalasins are utilized to study microfilament dynamics, cell cycle kinetics or chromosomal damage. Moreover, the polykaryon-inducing activity of cytochalasin D is suggested as a specific indicator for a P-gp-mediated multidrug-resistance phenotype and the reversing potency of chemosensitizers.