Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.     

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.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin.

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes

While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.     

A novel actin filament-capping protein from sea urchin eggs: a 20,000-molecular-weight protein-actin complex

A novel protein factor which reduced the low-shear viscosity of rabbit skeletal muscle actin was purified from a 0.6 M KCl-extract of an insoluble fraction of sea urchin eggs by ammonium sulfate fractionation, gel filtration column chromatography, DNase I column chromatography, and hydroxylapatite column chromatography. This protein factor was shown to be a one-to-one complex of a 20,000-molecular-weight protein and egg actin. This protein complex accelerated the initial rate of actin polymerization, but reduced the steady-state viscosity of F-actin. It inhibited at substoichiometric amounts the elongation of actin filaments on sonicated F-actin fragments and depolymerization of F-actin induced by dilution. In addition, it increased the critical concentration of actin for polymerization. All these effects of this protein complex on actin could be explained by the "capping the barbed end" of the actin filament by the complex. The 20,000-molecular-weight protein which was separated from actin also possessed the barbed end-capping activities, but differed from the complex in that it did not accelerate the polymerization of actin.     

Regulation of oscillations in filamentous actin content in polymorphonuclear leukocytes stimulated with leukotriene B(4) and platelet-activating factor.

Stimulation of neutrophils with LTB(4) or PAF results in the production of a rapidly oscillating actin polymerization/depolymerization response. Treatment of neutrophils with inhibitors of PKC prior to stimulation with ligand resulted in a masking of the F-actin oscillations. Because myosin has been shown to be a substrate for neutrophil PKC, this protein was investigated as a potential downstream mediator of F-actin oscillations. Stimulation of neutrophils with LTB(4) resulted in myosin light chain being serine phosphorylated in a PKC-dependent manner. This phosphorylation was shown to occur in a manner that is kinetically distinct from the myosin phosphorylation induced by FMLP, a potent activator of actin polymerization that alone does not induce F-actin oscillations. Additionally, disruption of intracellular actin-myosin interactions resulted in inhibition of LTB(4)- as well as PAF-induced F-actin oscillations. These data suggest that PKC and downstream phosphorylation of myosin as well as actin-myosin interaction may play roles in mediating the production of neutrophil F-actin oscillations.     

Purification and characterization of aginactin, a newly identified agonist-regulated actin-capping protein from Dictyostelium amoebae.

Amoeboid chemotaxis involves a regulated increase in actin nucleation activity that is correlated with an increase in actin polymerization occurring seconds after chemotactic stimulation (Carson, M., Weber, A., and Zigmond, S. H. (1986) J. Cell Biol. 103, 2707-2714; Hall, A. L., Warren, V., Dharmawardhane, S., and Condeelis, J. (1989) J. Cell Biol. 109, 2207-2213). We report the isolation and characterization of an agonist-regulated capping protein, aginactin, from Dictyostelium that may regulate these changes in actin nucleation activity. Aginactin is isolated from low speed supernatants of starved amoebae by sequential anion exchange, hydrophobic interaction, fast protein liquid chromatography anion exchange, and hydroxyapatite chromatography. Aginactin migrates with an apparent molecular weight of 70,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and gel filtration columns, suggesting that it is a globular monomer. Aginactin is a barbed-end capping protein by several criteria. It inhibits the rate and final extent of actin polymerization and increases the apparent critical concentration at substoichiometric ratios to actin. It also inhibits depolymerization of F-actin and inhibits polymerization at the barbed end of Limulus acrosomal bundles. Aginactin is unaffected by micromolar Ca2+, and it neither severs F-actin nor nucleates actin polymerization in either the presence or absence of Ca2+. Aginactin binds to and cosediments with F-actin and has an apparent Kd for capping F-actin of 2.7 nM.     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly.

The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.     

Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo.     

Mobile actin clusters and traveling waves in cells recovering from actin depolymerization

At the leading edge of a motile cell, actin polymerizes in close apposition to the plasma membrane. Here we ask how the machinery for force generation at a leading edge is established de novo after the global depolymerization of actin. The depolymerization is accomplished by latrunculin A, and the reorganization of actin upon removal of the drug is visualized in Dictyostelium cells by total internal reflection fluorescence microscopy. The actin filament system is reorganized in three steps. First, F-actin assembles into globular complexes that move along the bottom surface of the cells at velocities up to 10 microm/min. These clusters are transient structures that eventually disassemble, fuse, or divide. In a second step, clusters merge into a contiguous zone at the cell border that spreads and gives rise to actin waves traveling on a planar membrane. Finally, normal cell shape and motility are resumed. These data show that the initiation of actin polymerization is separated in Dictyostelium from front protrusion, and that the coupling of polymerization to protrusion is a later step in the reconstitution of a leading edge.     

pH control of actin polymerization by cofilin

Cofilin, a 21,000 molecular weight actin-regulatory protein (Nishida, E., Maekawa, S., and Sakai, H. (1984) Biochemistry 23, 5307-5313), was here shown to be capable of reversibly controlling actin polymerization and depolymerization in a pH-sensitive manner. When cofilin was reacted with F-actin at different pH, the depolymerized actin concentration (= monomeric actin concentration) was higher at elevated pH. At pH less than 7.3, the monomeric actin concentrations did not exceed approximately 1 microM even in the presence of excess amounts of cofilin, whereas at pH greater than 7.3 it increased in proportion to the concentration of cofilin added, and complete depolymerization of F-actin occurred by the addition of an excess amount of cofilin. Moreover, in the presence of cofilin, rapid interconversion of monomeric and polymeric forms of actin can be induced by simply changing the pH of the medium. Thus, this study provides a new possible mechanism regulating actin polymerization, pH control.     

The induction of protrusion by neomycin in human glioma cells is correlated with a decrease followed by an increase in filamentous actin.

In this paper we describe an experimental investigation of the mechanism of motility of vertebrate cells. Human glioma cells were treated with neomycin, an inhibitor of the phosphatidylinositol cycle; and changes in cell motility and the cytoskeleton were examined by video, fluorescence, and scanning electron microscopy and by cytofluorometry. Neomycin stimulates a single protrusion of lamellipodia from the cell margin, which is correlated with an initial rapid decrease in the amount of F-actin throughout the cell, especially at the cell edge; the fragmentation of actin filaments within the lamellipodia; and the subsequent de novo polymerization of F-actin in a marginal band at the leading edge of lamellipodia. Changes in F-actin are paralleled by changes in the distribution and amount of gelsolin. These results support the hypothesis that protrusion is initiated by the gelsolin-mediated severing and subsequent depolymerization of cortical actin filaments, which weakens the cell cortex, allowing hydrostatic or gel osmotic pressure to force the cell margin to protrude. The accompanying polymerization of filaments actin at the leading edge of the protrusion may stabilize the protrusion and support its expansion.     

Binding of human angiogenin inhibits actin polymerization

Angiogenin is a potent inducer of angiogenesis, a process of blood vessel formation. It interacts with endothelial and other cells and elicits a wide range of cellular responses including migration, proliferation, and tube formation. One important target of angiogenin is endothelial cell-surface actin and their interaction might be one of essential steps in angiogenin-induced neovascularization. Based on earlier indications that angiogenin promotes actin polymerization, we studied the binding interactions between angiogenin and actin in a wide range of conditions. We showed that at subphysiological KCl concentrations, angiogenin does not promote, but instead inhibits polymerization by sequestering G-actin. At low KCl concentrations angiogenin induces formation of unstructured aggregates, which, as shown by NMR, may be caused by angiogenin’s propensity to form oligomers. Binding of angiogenin to preformed F-actin does not cause depolymerization of actin filaments though it causes their stiffening. Binding of tropomyosin and angiogenin to F-actin is not competitive at concentrations sufficient for saturation of actin filaments. These observations suggest that angiogenin may cause changes in the cell cytoskeleton by inhibiting polymerization of G-actin and changing the physical properties of F-actin.     

Passive mechanical behavior of human neutrophils: effects of colchicine and paclitaxel.

The role of microtubules in determining the mechanical rigidity of neutrophils was assessed. Neutrophils were treated with colchicine to disrupt microtubules, or with paclitaxel to promote formation of microtubules. Paclitaxel caused an increase in the number of microtubules in the cells as assessed by immunofluorescence, but it had no effect on the presence or organization of actin filaments or on cellular mechanical properties. Colchicine at concentrations <1.0 microM caused disruption of microtubular structures, but had little effect on either F-actin or on cellular mechanical properties. Higher concentrations of colchicine disrupted microtubular structure, but also caused increased actin polymerization and increases in cell rigidity. Treatment with 10 microM colchicine increased F-actin content by 17%, the characteristic cellular viscosity by 30%, the dependence of viscosity on shear rate by 10%, and the cortical tension by 18%. At 100 microM colchicine the corresponding increases were F-actin, 25%; characteristic viscosity, 50%; dependence of viscosity on shear rate, 20%; and cortical tension, 21%. These results indicate that microtubules have little influence on the mechanical properties of neutrophils, and that increases in cellular rigidity caused by high concentrations of colchicine are due to a secondary effect that triggers actin polymerization. This study supports the conclusion that actin filaments are the primary structural determinants of neutrophil mechanical properties.     

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.     

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.     

Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.     

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.     

Gamma-Actinin, a new regulatory protein from rabbit skeletal muscle. I. Purification and characterization.

A new regulatory protein which we have designated as gamma-actinin has been isolated from native thin filaments of rabbit skeletal muscle. Depolymerized native thin filaments were fractionated by salting out with ammonium sulfate, and the precipitates obtained at 40--60% ammonium sulfate saturation were further subjected to DEAE-Sephadex and Sephadex G-200 column chromatography. The purified gamma-actinin was shown to have a chain weight of 35,000 daltons and had a strong inhibitory action on the polymerization of G-actin. The results of amino acid analysis indicated a unique amino acid composition of gamma-actinin as compared with other structural proteins of muscle. Non-polar and neutral amino acid residues were abundant. One cysteine residue was contained per one molecule of gamma-actinin and played a critical role in the maintenance of the inhibitory activity. Pelleting of gamma-actinin with F-actin showed that gamma-actinin binds to F-action.