A possible complementary role of actin microfilaments and microtubules in triacylglycerol secretion by isolated rate hepatocytes

Incubation of isolated rat hepatocytes with phalloidin, cytochalasins (which, respectively, stabilize and destabilize actin microfilaments), or colchicine (which inhibits polymerization of microtubules), resulted in a dose-dependent inhibition of triacyglycerol secretion (an index of very low density lipoprotein secretion). Upon removal of drugs from incubation media, the inhibitory effect of cytochalasin D on triacylglycerol secretion was reversible, while such was not the case for phalloidin. When used at maximal concentrations, the combined presence of phalloidin + colchicine or cytochalasin D + colchicine had additive inhibitory effects upon hepatic triacylglycerol secretion, which was virtually blocked; this was not the case for phalloidin + cytochalasin D. These experiments support the concept that microfilaments and microtubules may have complementary functions for the hepatic secretion of very low density lipoproteins.     

Influence of phalloidin on ATP hydrolysis during actin polymerization

The influence of phalloidin on the ATP hydrolysis associated with actin polymerization was investigated. Whereas in the absence of phalloidin actin-bound ATP was totally hydrolyzed during polymerization, ATP hydrolysis was not complete after actin polymerization in the presence of phalloidin: 5-10% of ATP remained unhydrolyzed and disappeared only after 2 days.     

Actin polymerization modifies stimulus-oxidase coupling in rat neutrophils

Oxidase activity in rat neutrophils was monitored by oxygen consumption rate and luminol-dependent chemiluminescence. Two agents which inhibit actin polymerization, cytochalasin B and dihydrocytochalasin B, produced a marked enhancement (up to 10-fold) of oxidase activation induced by two Ca2+-dependent stimuli, chemotactic peptide and ionophore A23187. In contrast, activation by the calcium-independent stimulus, phorbol myristate acetate, was unaffected by these agents. Other agents that interact with the cytoskeleton, phalloidin and colchicine have no effect on activation by any stimulus tested. The effect of cytochalasin B, when added after stimulation by chemotactic peptide, was transient with t0.5 approx. 10 s. Similarly, the degree of actin polymerization following stimulation by chemotactic peptide was transient, decaying with a t0.5 of approx. 10 s. The half-maximal concentration of cytochalasin B for inhibition of actin polymerization was similar to that for enhancement of oxidase activation. It was concluded, therefore, that the intracellular Ca2+ rise in rat neutrophils that accompanies stimulation by chemotactic peptide affects actin polymerization in a manner that modifies oxidase activation.     

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 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.     

Actin microfilaments, cell shape, and secretory processes in isolated rat hepatocytes. Effect of phalloidin and cytochalasin D

The effects of phalloidin and cytochalasin D, drugs which, respectively, stabilize and destabilize actin microfilaments, have been tested on isolated rat hepatocytes. Both drugs produced a modification of cell shape, characterized by protrusions bulging from the cytoplasm. In phalloidin-treated hepatocytes, an accumulation of actin microfilamentous network was detectable at the base of each protrusion by electron microscopy, immunofluorescence, and HMM decoration. This accumulation of microfilaments was absent in cytochalasin D-treated cells. The release of triglycerides, an index of very low density lipoprotein secretion, was inhibited by phalloidin or cytochalasin D, and accompanied by an increase in cellular triglycerides. At the electron microscope examination, triglyceride accumulation was represented by fat droplets and vesicle-enclosed, very low density lipoprotein-like particles. Total protein and albumin secretion was only very slightly modified by either one of these drugs. With the use of various phalloidin analogs, a correlation was observed between their respective ability to stabilize F-actin in vitro, and their effects on cell shape and triglyceride secretion. In conclusion, phalloidin, and cytochalasin D: (a) modify the shape of isolated hepatocytes; (b) inhibit lipoprotein secretion. These effects possibly result from a modification of actin microfilament function.     

Rapid microfilament reorganization induced in isolated rat hepatocytes by microcystin-LR, a cyclic peptide toxin

The cyclic heptapeptide hepatotoxin microcystin-LR from the cyanobacterium Microcystis aeruginosa induces rapid and characteristic deformation of isolated rat hepatocytes. We investigated the mechanism(s) responsible for cell shape changes (blebbing). Our results show that the onset of blebbing was accompanied neither by alteration in intracellular thiol and Ca2+ homeostasis nor by ATP depletion. The irreversible effects were insensitive to protease and phospholipase inhibitors and also to thiol-reducing agents, excluding the involvement of enhanced proteolysis, phospholipid hydrolysis, and thiol modification in microcystin-induced blebbing. In contrast, the cell shape changes were associated with a remarkable reorganization of microfilaments as visualized both by electron microscopy and by fluorescent staining of actin with rhodamine-conjugated phalloidin. The morphological effects and the microfilament reorganization were specific for microcystin-LR and could not be induced by the microfilament-modifying drugs cytochalasin D or phalloidin. Using inhibition of deoxyribonuclease I as an assay for monomeric actin, we found that the microcystin-induced reorganization of hepatocyte microfilaments was not due to actin polymerization. On the basis of the rapid microfilament reorganization and the specificity of the effects, it is suggested that microcystin-LR constitutes a novel microfilament-perturbing drug with features that are clearly different from those of cytochalasin D and phalloidin.     

The small GTP-binding proteins, Rac and Rho, regulate cytoskeletal organization and exocytosis in mast cells by parallel pathways.

In mast cells, activation of GTP-binding proteins induces centripetal reorganization of actin filaments. This effect is due to disassembly, relocalization, and polymerization of F-actin and is dependent on two small GTPases, Rac and Rho. Activities of Rac and Rho are also essential for the secretory function of mast cells. In response to GTP-gamma-S and/or calcium, only a proportion of permeabilized mast cells is capable of secretory response. Here, we have compared actin organization of secreting and nonsecreting cell populations. We show that the cytoskeletal and secretory responses are strongly correlated, indicating a common upstream regulator of the two functions. The secreting cell population preferentially displays both relocalization and polymerization of actin. However, when actin relocalization or polymerization is inhibited by phalloidin or cytochalasin, respectively, secretion is unaffected. Moreover, the ability of the constitutively active mutants of Rac and Rho to enhance secretion is also unaffected in the presence of cytochalasin. Therefore, Rac and Rho control these two functions by divergent, parallel signaling pathways. Cortical actin disassembly occurs in both secreting and nonsecreting populations and does not, by itself, induce exocytosis. A model for the control of exocytosis is proposed that includes at least four GTP-binding proteins and suggests the presence of both shared and divergent signaling pathways from Rac and Rho.     

Effects of phalloidin and cytochalasin B on cytoskeletal structures in cultured rat hepatocytes

Summary In short-term cultures of rat hepatocytes, bile canaliculi enclosed between unseparated cell couplets are able to perform periodical contractions resulting in expulsion of bile. Pericanalicular cytoskeletal proteins are involved in canalicular contractility: F-actin, myosin and tropomyosin are associated around bile canaliculi, as revealed by staining with tetramethylrhodaminyl-phalloidin and by immunofluorescence. Bile canalicular contractility is distributed by cholestatic agents that are known to interfere with actin polymerization; e.g., phalloidin and also cytochalasin B inhibit canalicular contractility and cause pericanalicular vacuolization and formation of blebs. Whereas the association of the cytoskeletal proteins is not affected by treatment with cytochalasin B, treatment with phalloidin results in dissociation of F-actin and myosin, indicating that binding of phalloidin to F-actin impairs its molecular interaction with myosin.     

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.     

Intracellular pH modulates taste receptor cell volume and the phasic part of the chorda tympani response to acids

The relationship between cell volume and the neural response to acidic stimuli was investigated by simultaneous measurements of intracellular pH (pHi) and cell volume in polarized fungiform taste receptor cells (TRCs) using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in vitro and by rat chorda tympani (CT) nerve recordings in vivo. CT responses to HCl and CO2 were recorded in the presence of 1 M mannitol and specific probes for filamentous (F) actin (phalloidin) and monomeric (G) actin (cytochalasin B) under lingual voltage clamp. Acidic stimuli reversibly decrease TRC pHi and cell volume. In isolated TRCs F-actin and G-actin were labeled with rhodamine phalloidin and bovine pancreatic deoxyribonuclease-1 conjugated with Alexa Fluor 488, respectively. A decrease in pHi shifted the equilibrium from F-actin to G-actin. Treatment with phalloidin or cytochalasin B attenuated the magnitude of the pHi-induced decrease in TRC volume. The phasic part of the CT response to HCl or CO2 was significantly decreased by preshrinking TRCs with hypertonic mannitol and lingual application of 1.2 mM phalloidin or 20 microM cytochalasin B with no effect on the tonic part of the CT response. In TRCs first treated with cytochalasin B, the decrease in the magnitude of the phasic response to acidic stimuli was reversed by phalloidin treatment. The pHi-induced decrease in TRC volume induced a flufenamic acid-sensitive nonselective basolateral cation conductance. Channel activity was enhanced at positive lingual clamp voltages. Lingual application of flufenamic acid decreased the magnitude of the phasic part of the CT response to HCl and CO2. Flufenamic acid and hypertonic mannitol were additive in inhibiting the phasic response. We conclude that a decrease in pHi induces TRC shrinkage through its effect on the actin cytoskeleton and activates a flufenamic acid-sensitive basolateral cation conductance that is involved in eliciting the phasic part of the CT response to acidic stimuli.     

Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate

The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function.     

Comparative studies on the energetics of platelet responses induced by different agonists.

The correlation between energy consumption and platelet responses induced by collagen, A23187 and ADP was investigated and compared with the energetics of thrombin-stimulated platelets established in earlier work. Aggregation, measured as single-platelet disappearance, and secretion correlated quantitatively with the increment but not with the total consumption of energy, suggesting that the former reflects the energy cost of these responses. The cost of complete aggregation was 2-3 mumol of ATP equivalents/10(11) platelets with collagen, ADP and thrombin as the stimulus. The cost of complete dense-granule secretion was 0.5-0.8 mumol of ATP equivalents/10(11) platelets with all agonists tested. The cost of combined secretion of alpha-granule and acid hydrolase granule contents was 5-7 mumol of ATP equivalents/10(11) platelets with thrombin and collagen. However, in the presence of A23187 much more energy was consumed during aggregation and secretion. Also ADP triggered more energy consumption during secretion than was seen with the other inducers. The effect of inhibitors of aggregation and secretion was investigated in thrombin-stimulated platelets. Raising the cellular cyclic AMP content sharply decreased the increment in energy consumption as well as aggregation and secretion. The cytoskeleton-disrupting agents cytochalasin B and colchicine left the increment in energy consumption intact, but decreased the basal consumption seen in unstimulated platelets. This was accompanied by normal (cytochalasin B) or diminished (colchicine) aggregation and secretion. Apart from the latter exception, all inhibitors decreased secretion and incremental energy consumption in parallel, thereby preserving the energy-versus-secretion relationship established in earlier work. In contrast, aggregation and energy consumption varied independently, suggesting that the coupling with energy consumption is much weaker for this response.     

Role of the cytoskeleton in secretion of chloride by shark rectal gland

The salt gland of the spiny dogfish, Squalus acanthias, can be stimulated to secrete chloride by two different endogenous peptides: cardiac natriuretic peptide (CNP) and the neurotransmitter, vasoactive intestinal peptide (VIP). We examined the role of the actin cytoskeleton and of myosin light chains in this process by perfusing isolated rectal glands with and without an inhibitor of actin filament organization (cytochalasin D) and an inhibitor of myosin light chain kinase (ML-7). Cytochalasin D, 10(-6) M, reduced secretion stimulated by a 1-min bolus of CNP (5x10(-7) M) by 50-60%. In the presence of 10(-2) M procaine (which blocks neural release of VIP), cytochalasin D completely prevented CNP stimulation. In contrast, cytochalasin D did not inhibit stimulation of the rectal gland by VIP or by forskolin. Similarly, 5x10(-6)M ML-7 almost completely inhibited direct stimulation of rectal gland secretion by CNP, but did not alter chloride secretion induced by VIP or forskolin. Finally, the average time between hormonal injection and activation of secretion was 2 min longer for CNP than for VIP, consistent with the hypothesis that a contractile cellular function involving the cytoskeleton is important in CNP-induced chloride secretion, but less so when secretion is stimulated by VIP.     

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(α-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.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

Actin polymerization. The mechanism of action of cytochalasin D

Fluorescence changes using actin covalently labeled with N-(1-pyrenyl)iodoacetamide have been used to determine the effect of cytochalasin D on actin polymerization. A mechanism for the effect of cytochalasin D on actin polymerization is presented, which explains the experimental observation of a cytochalasin D-induced increase in the initial rate of polymerization and a decrease in the final extent of the reaction. Central to this mechanism is the Mg2+-dependent formation of cytochalasin D-induced dimers. The dimers serve as nuclei to enhance the polymerization rate. Binding of Mg2+ to a low affinity site on the dimer induces a conformational change which can be observed as a rapid fluorescence increase. A subsequent time-dependent fluorescence decrease observed prior to polymerization appears to represent ATP hydrolysis resulting in dissociation of the dimer and release of actin monomers containing ADP. We postulate that a slow rate of exchange of ATP for bound ADP relative to hydrolysis results in the accumulation of monomers containing ADP. As these monomers have a high critical concentration, the final extent of polymerization is reduced dramatically. The Mg2+ dependence of the final extent of polymerization in the presence of cytochalasin D is also explained in the context of this mechanism.     

Actin Turnover-Mediated Gravity Response in Maize Root Apices: Gravitropism of Decapped Roots Implicates Gravisensing Outside of the Root Cap

The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap.Key Words: actin cytoskeleton, gravisensing, graviresponding, root cap     

Role of actin microfilaments in depression of acetylcholine-induced current in Helix lucorum neurons on cellular analogue of habituation

Toxins that impair the function of actin microfilaments in cytoskeleton, cytochalasin B (disrupts microfilaments by inhibiting actin polymerization) and phalloidin (binds polymeric F-actin, stabilizing it and interfering with the function of actin-rich structures) reduce the depression of acetylcholine-induced inward current in Helix lucorum command neurons of defensive behavior during rhythmical local acetylcholine applications to soma (cellular analogue of habituation). These results and mathematical simulation allow us to suggest that the depression of cholinosensitivity of extrasynaptic membrane zones in command neurons on the cellular analogue of habituation is associated with the involvement of actin microfilaments in reduction of the number of membrane cholinoreceptors.     

Mechanism of action of phalloidin on the polymerization of muscle actin

Under conditions where muscle actin only partially polymerizes, or where it does not polymerize at all, a significant enhancement of polymerization was observed if equimolar phalloidin was also present. The increased extent of polymerization in the the presence of phalloidin can be explained by the reduced critical actin concentration of partially polymerized populations at equilibrium. Under such conditions, the rate of polymerization, as judged by the length of time to reach half the viscosity plateau, was found to be essentially independent of the phalloidin concentration. Moreover, the initial rate of polymerization of actin was also found to be independent of phalloidin concentration. However, phalloidin apparently causes a reduction in the magnitude of the reverse rates in the polymerization reaction, as was demonstrated by the lack of depolymerization of phalloidin-treated actin polymers. This effect of phalloidin is also supported by the identification of actin nuclei and short polymers in populations of G-actin incubated with phalloidin in the absence of added KCl. Our conclusion, then, is that phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed.