Reversal of profilin inhibition of actin polymerization invitro by erythrocyte cytochalasin-binding complexes and cross-linked actin nuclei

Actin polymerization in 2 mM MgCl₂ is known to be inhibited by profilin. We found that small amounts of cytochalasin-binding complexes from human red cell membranes or actin nuclei cross-linked by $\text{p}\text{-}\text{NN′}$-phenylenebismaleimide can reverse the inhibitory action of profilin, leading to the rapid polymerization of the actin. This type of polymerization is inhibited by low concentrations of cytochalasin B. These results indicate that (a) the complexes and nuclei promote actin polymerization in the presence of profilin by providing sites onto which actin monomers can be added, and (b) profilin and cytochalasin B affect two distinct steps (i.e. nucleus formation and filament elongation, respectively) in the polymerization reaction.     

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.     

Mechanism of action of cytochalasin B on actin

Substoichiometric cytochalasin B (CB) inhibits both the rate of actin polymerization and the interaction of actin filaments in solution. The polymerization rate is reduced by inhibition of actin monomer addition to the "barbed" end of the filaments where monomers normally add more rapidly. 2 microM CB reduces the polymerization rate by up to 90%, but has little effect on the rate of monomer addition at the slow ("pointed") end of the filaments and no effect on the rate of filament annealing. Under most ionic conditions tested, 2 microM CB reduces the steady state high shear viscosity by 10-20% and increases the steady state monomer concentration by a factor of 2.5 or less. In addition to the effects on the polymerization process, 2 microM CB strongly reduces the low shear viscosity of actin filaments alone and actin filaments cross-linked by a variety of macromolecules. This may be due to inhibition of actin filament-filament interactions which normally contribute to network formation. Since the inhibition of monomer addition and of actin filament network formation have approximately the same CB concentration dependence, a common CB binding site, probably the barbed end of the filament, may be responsible for both effects.     

Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.     

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

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

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.     

Actin assembly activity of cytochalasins and cytochalasin analogs assayed using fluorescence photobleaching recovery

The effects on actin self-assembly of 9 common cytochalasins and 9 synthetic analogs have been assayed using fluorescence photobleaching recovery (FPR). The specific assembly activities of cytochalasins determined by this assay are (i) reduction of the fraction of actin molecules incorporated into filaments; (ii) increase of the steady-state diffusion coefficients of filaments, from which filaments shortening may be inferred; and (iii) acceleration of the initial rate of assembly. Of the compounds studied, only cytochalasin D shows strong activity of all three types. The range of activities shown by other compounds indicates clearly that these three activity types are distinct and independent. Inspection of the molecular structures of these 18 compounds for correlation of structure and activity reveals that the three different activities depend on distinct structural features. The Mg2+ dependence of filament-shortening activity by certain cytochalasins may be explained by the Mg2+ chelating ability of two suitably positioned oxygen atoms on the convex face of the bicyclic isoindolone system. Inhibition of filament elongation may involve very specific, high-affinity cytochalasin interactions at a binding site on terminal actin molecules, while accelerating activity may occur by weaker, less specific binding interactions of cytochalasins with monomeric actin.     

Comparative study on effects of cytochalasins B and D on F-actin content in different cell lines and different culture conditions.

Effects of cytochalasins on actin polymerization state in living cells were measured using fluorimetry of TRITC-phalloidin bound to F-actin. Normal (3T3) and tumour (SV-3T3, B16 melanoma, and Ehrlich ascites) cells were treated with cytochalasin B and cytochalasin D (1 microgram/ml). Three effects of cytochalasins were demonstrated--depolymerization of F-actin, promotion of polymerization, and redistribution of actin without change in polymerization state. Occurrence of a given effect was dependent on cell type, cell density, cytochalasin concentration and type. This indicates that cells from different lines, and even the same cells in different culture conditions may differ significantly in their state of actin polymerization, which we suppose is the cause of their different reactions to cytochalasins. Accordingly, caution should be taken in generalizing the results concerning the effect of cytochalasis on the polymerization state of actin.     

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.     

Length distribution of F-actin in Dictyostelium discoideum

Inhibition of deoxyribonuclease I activity was used to assay the actin monomers and the pointed ends of actin filaments in lysates of Dictyostelium discoideum. The KD for the binding reaction was 0.2-0.3 nM. Total cellular actin was 93 microM in monomers (approximately 0.1 fmol/cell) of which roughly half was initially polymeric. Essentially all of the filamentous actin (F-actin) was readily pelleted in the microcentrifuge and was therefore presumed to be in the cytoskeleton. Free F-actin barbed ends, measured as pelletable [3H]cytochalasin B, numbered 1.8 x 10(5)/cell; nuclei for the polymerization of rabbit muscle globular (monomeric) actin numbered 2.0 x 10(5)/cell; and pointed ends, determined by their inhibition of deoxyribonuclease I, numbered 3.6 x 10(5)/cell. These values suggest that half the barbed ends might be occluded. On average, the filaments contained approximately 76 subunits and were therefore about 0.2 micron long. The distribution of their lengths was estimated from the time course of depolymerization following vast dilution. Three populations were defined. In one experiment, the smallest population contained 71% of the F-actin mass and 96% of the pointed ends; these filaments averaged 80 subunits or 0.22 microns in length. An intermediate population contained 14% of the F-actin mass and 3% of the filaments; these were roughly 460 subunits (1.3 microns) long. The largest population contained 15% of the F-actin mass in about 0.3% of the filaments; these were 13 microns in length, about the diameter of the cell. The numerous short filaments might populate a cortical mesh, while the long filaments might constitute endoplasmic bundles.     

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.     

Cytochalasin inhibits the rate of elongation of actin filament fragments

Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B, which is in keeping with the effects that have been reported on cell motility and with binding to a class of high-affinity binding sites from human erythrocyte membranes (Lin and Lin. 1978. J. Biol. CHem. 253:1415; Lin and Lin. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:2345); 5x10(-7) M cytochalasin B lowers it to 70 percent of the control value, whereas 10(-7) M cytochalasin B lowers the rate to 25 percent. Fragments of F-actin were used to increase the rate of assembly fivefold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2x10(-7) M and 10(-8) M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4x10(-8) M and 1x10(-9) M, respectively. A scatchard plot has been obtained using [(3)H]cytochalasin B binding to F-actin in the absence of ATP. The K(d) from this plot (approximately 4x10(-8) M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly one per F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends.     

Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1- pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre- activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.     

Association of Serotonin, Dopamine, or Noradrenaline with an Actin-Like Component in Pheochromocytoma (PC12) Cells

A rat pheochromocytoma (PC12) cell line was used to examine the possibility that 5-hydroxytryptamine (serotonin), 3,4-dihydroxyphenylethylamine (dopamine), or noradrenaline may be associated with cytoplasmic actin, as was suggested by previous in vitro binding studies on an actin-like protein from rat brain synaptosomes. When PC12 cells were incubated with [3H]serotonin. [3H]dopamine, or [3H]noradrenaline for 30 min at 37 degrees C, approximately 2-4% of the radioactivity present in the cells was found to be associated with a high-molecular-weight (actin-like) component in supernatant fractions. Evidence relating this monoamine binding component to actin filaments includes: (a) its strong absorption by myosin filaments at low ionic strength: (b) a decrease in its affinity for myosin in the presence of 1 mM ATP, which lowers the affinity of authentic actin for myosin: (c) displacement of bound [3H]serotonin from it by DNase I, which binds strongly to actin and which inhibits [3H]serotonin binding to actin in vitro; (d) an increase in its binding of each monoamine (by 25-40%) after PC12 cells were preincubated with 10 microM cytochalasin B (a drug that induces depolymerization of F-actin). These findings suggest that serotonin, dopamine, or noradrenaline may associate with actin filaments in vivo.     

Mechanism of action of cytochalasin: evidence that it binds to actin filament ends

To test the idea that cytochalasin retards actin assembly by binding to filament ends, we have designed a new assay for cytochalasin binding in which the number of filament ends can be varied independently of the total actin concentration. Actin is reacted with polylysine-coated polystyrene beads to make filament ends (Brown and Spudich, 1979, J. Cell Biol. 80:499-504) and then reacted with [3H]cytochalasin B. We have found that cytochalasin B binds to beads in the presence of actin, and that the number of cytochalasin B binding sites can be varied as a function of the number of filament ends independent of the total actin concentration by varying the bead concentration.     

Evidence that forskolin binds to the glucose transporter of human erythrocytes

Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.     

Polycation induced actin bundles

Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4 Å) MTS-1 (1,1-Methanedyl Bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.     

Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.     

Wide-ranging effects of eight cytochalasins and latrunculin A and B on intracellular motility and actin filament reorganization in characean internodal cells

Numerous forms of cytochalasins have been identified and, although they share common biological activity, they may differ considerably in potency. We investigated the effects of cytochalasins A, B, C, D, E, H and J and dihydrocytochalasin B in an ideal experimental system for cell motility, the giant internodal cells of the characean alga Nitella pseudoflabellata. Cytochalasins D (60 microM) and H (30 microM) were found to be most suited for fast and reversible inhibition of actin-based motility, while cytochalasins A and E arrested streaming at lower concentrations but irreversibly. We observed no clear correlation between the ability of cytochalasins to inhibit motility and the actual disruption of the subcortical actin bundle tracks on which myosin-dependent motility occurs. Indeed, the actin bundles remained intact at the time of streaming cessation and disassembled only after one to several days' treatment. Even when applied at concentrations lower than that required to inhibit cytoplasmic streaming, all of the cytochalasins induced reorganization of the more labile cortical actin filaments into actin patches, swirling clusters or short rods. Latrunculins A and B arrested streaming only after disrupting the subcortical actin bundles, a process requiring relatively high concentrations (200 microM) and very long treatment periods of >1 d. Latrunculins, however, worked synergistically with cytochalasins. A 1 h treatment with 15 nM latrunculin A and 4 microM cytochalasin D induced reversible fragmentation of subcortical actin bundles and arrested cytoplasmic streaming. Our findings provide insights into the mechanisms by which cytochalasins and latrunculins interfere with characean actin to inhibit motility.     

Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative

The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.