Actomyosin interactions with insulin-storage granules in vitro.

Interactions between actomyosin and insulin storage granules isolated from rat islets of Langerhans have been examined in a simple system in vitro, which allows comparison of the sedimentation of the granules in the presence of absence of actomyosin in various conditions. Actomyosin altered granule-sedimentation rates in a manner consistent with the binding of the granules of actomyosin filaments. This interaction was enhanced by addition of ATP (1.5 mM) but unaltered by addition of CaCl2, by calmodulin or by calmodulin in the presence of 10 microM-CaCl2. Addition of EGTA (0.1 mM), cyclic AMP (10 microM) of cytochalasin B (10 microgram/ml) were also without effects in these conditions. Pre-incubation of granules with phospholipase c did not affect granule-actomyosin interaction. Ultrastructural studies showed close contacts between the membranes of the granules and actomyosin filaments. The results indicate the possibility that actomyosin might provide the motile force for granule translocation during the insulin secretory process.     

Muscle actin filaments bind pituitary secretory granules in vitro

Hog anterior pituitary secretory granules sediment at 3,000 g. When rat or rabbit skeletal muscle actin filaments are present with the granules, the sedimentation decreases markedly. Depolymerized actin or viscous solutions of Ficoll and collagen have no effect on granule sedimentation. With this assay, actin filaments bind secretory granules (consisting of the proteinaceous core plus limiting membrane), secretory granule membranes, mitochondria, artificial lecithin liposomes, and styrene-butadiene microspheres, but have little or no interaction with membrane-free secretory granule cores and albumin microspheres. A secretory granule-actin complex sedimentable between 3,000 g and 25,000 g can be isolated. Metal ions, nucleotides, salts, dithiothreitol, or pretreatment of the granules with trypsin do not destroy the binding, which appears to be a lipophilic interaction.     

Polymerization of actin induced by a molar excess of ATP in a low salt buffer

The polymerization of actin induced by dilution has previously been reported, where a 1000-fold molar excess of ATP over actin resulted when actin was diluted to 4.0 microg/ml in low salt buffer A (0.1 mM ATP, 0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 5 mM 2-mercaptoethanol, 1 mM NaN3). Filaments formed by the addition of ATP to a 1000-fold molar excess over actin in buffer B (0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 1 mM NaN3) were then separated by gel-filtration. When ATP was removed from these filaments using Dowex-1, depolymerization occurred. Thus, the reversible polymerization induced by the dilution of actin or by addition of ATP can be ascribed to the binding of ATP at the low affinity site of actin.     

Polymerization of actin induced by a molar excess of ATP in a low salt buffer.

The polymerization of actin induced by dilution has previously been reported, where a 1000-fold molar excess of ATP over actin resulted when actin was diluted to 4.0 micrograms/ml in low salt buffer A (0.1 mM ATP, 0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 5 mM 2-mercaptoethanol, 1 mM NaN3). Filaments formed by the addition of ATP to a 1000-fold molar excess over actin in buffer B (0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 1 mM NaN3) were then separated by gel-filtration. When ATP was removed from these filaments using Dowex-1, depolymerization occurred. Thus, the reversible polymerization induced by the dilution of actin or by addition of ATP can be ascribed to the binding of ATP at the low affinity site of actin.     

Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin.

A specific association between spectrin and the inner surface of the human erythrocyte membrane has been examined by measuring the binding of purified [32P]spectrin to inside out, spectrin-depleted vesicles and to right side out ghost vesicles. Spectrin was labeled by incubating erythrocytes with 32Pi, and eluted from the ghost membranes by extraction in 0.3 mM NaPO4, pH 7.6. [32P]Spectrin was separated from actin and other proteins and isolated in a nonaggregated state as a So20,w = 7 S (in 0.3 mM NaPO4) or So20,w = 8 S (in 20 mM KCl, 0.3 mM NaPO4) protein after sedimentation on linear sucrose gradients. Binding of [32P]spectrin to inverted vesicles devoid of spectrin and actin was at least 10-fold greater than to right side out membranes, and exhibited different properties. Association with inside out vesicles was slow, was decreased to the value for right side out vesicles at high pH, or after heating spectrin above 50 degrees prior to assay, and was saturable with increasing levels of spectrin. Binding to everted vesicles was rapid, unaffected by pH or by heating spectrin, and rose linearly with the concentration of spectrin. Scatchard plots of binding to inverted vesicles were linear at pH 7.6, with a KD of 45 microng/ml, while at pH 6.6, plots were curvilinear and consistent with two types of interactions with a KD of 4 and 19 microng/ml, respectively. The maximal binding capacity at both pH values was about 200 microng of spectrin/mg of membrane protein. Unlabeled spectrin competed for binding with 50% displacement at 27 microng/ml. [32P]Spectrin dissociated and associated with inverted vesicles with an identical dependence on ionic strength as observed for elution of native spectrin from ghosts. MgCl2, CaCl2 (1 to 4 mM) and EDTA (0.5 to 1 mM) had little effect on binding in the presence of 20 mM KCl, while at low ionic strength, MgCl2 (1 mM) increased binding and inhibited dissociation to the same extent as 10 to 20 mM KCl. Binding was abolished by pretreatment of vesicles with 0.1 M acetic acid, or with 0.1 microng/ml of trypsin. The periodic acid-Schiff-staining bands were unaffected by trypsin digestion which destroyed binding; mild digestion, which decreased binding only 50%, converted Band 3 almost completely to a membrane-bound 50,000-dalton fragment resistant to further proteolysis. These experiments suggest that attachment of spectrin to the cytoplasmic surface of the membrane results from a selective protein-protein interaction which is independent of erythrocyte actin. A direct role of the major sialoglycoprotein or Band 3 as a membrane binding site appears unlikely.     

Opposite effects of alfa-actinin and of fructose 1,6-bisphosphate aldolase on the microfilament network. The role of orthophosphate revisited

At pH 7.5, in the presence of 0.1 M KCl, 2 mM MgCl2 and 15 mM phosphate, the binding of 1 molecule of alfa-actinin for each strand of 1000 actin monomers doubles the apparent viscosity of an F-actin solution (12 microM as the monomer). Further binding of one molecule of aldolase for each strand of 280 actin monomers halves the apparent viscosity of the alfa-actinin-F-actin system without any desorption of alfa-actinin. The effect of aldolase is not hindered by the addition of 0.1 mM fructose 1,6-bisphosphate. It is shown that orthophosphate acts as a damper of the regulatory effect of fructose bisphosphate on the interaction between aldolase and microfilaments.     

Reinvestigation of the inhibition of actin polymerization by profilin

In buffer containing 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 5 mM imidazole, pH 7.5, 0.1 mM CaCl2, 0.2 mM dithiothreitol, 0.01% NaN3, and 0.2 mM ATP, the KD for the formation of the 1:1 complex between Acanthamoeba actin and Acanthamoeba profilin was about 5 microM. When the actin was modified by addition of a pyrenyl group to cysteine 374, the KD increased to about 40 microM but the critical concentration (0.16 microM) was unchanged. The very much lower affinity of profilin for modified actin explains the anomalous critical concentrations curves obtained for 5-10% pyrenyl-labeled actin in the presence of profilin and the apparently weak inhibition by profilin of the rate of filament elongation when polymerization is quantified by the increase in fluorescence of pyrenyl-labeled actin. Light-scattering assays of the polymerization of unmodified actin in the absence and presence of profilin gave a similar value for the KD (about 5-10 microM) when determined by the increase in the apparent critical concentration of F-actin at steady state at all concentrations of actin up to 20 microM and by the inhibition of the initial rates of polymerization of actin nucleated by either F-actin or covalently cross-linked actin dimer. In the same buffer, but with ADP instead of ATP, the critical concentration of actin was higher (4.9 microM) and the KD of the profilin-actin complex was lower for both unmodified (1-2 microM) and 100% pyrenyl-labeled actin (4.9 microM).     

Ca2+-calmodulin-dependent polymerization of actin by myelin basic protein

The interaction between myelin basic protein (MBP) and G-actin was studied under nonpolymerizing conditions, i.e.,2mM HEPES, pH 7.5, 0.1 mM CaCl2 and 0.2 mM ATP. Fluorescence studies using pyrenyl-actin and the measurements of ATP hydrolysis rate show that MBP induces changes in the structure of the actin monomer similar to those occurring during polymerization by salt. Electron microscope observations of the MBP-G-actin complex reveal the presence of filamentous structures which appear as separate filaments or as bundles of filaments in lateral association. These filaments are polar as visualized by attachment of heavy meromyosin. The biochemical data together with electron microscope observations suggest that the binding of MBP to G-actin under non-polymerizing conditions induces an interaction between actin monomers leading to the formation of filamentous structures which may be similar to F-actin filaments. The effects of MBP on G-actin can be reversed by calmodulin in the presence of Ca2+.     

Transformation of actin-encapsulating liposomes induced by cytochalasin D.

Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed either disk or dumbbell shape, but when cytochalasin D was added to the medium surrounding the liposomes, they were found to become spindle shaped. Liposomes containing bovine serum albumin that were given cytochalasin D and actin-containing liposomes that were given dimethylformamide, the solvent for cytochalasin D, did not transform. These results indicated actin-cytochalasin interaction is involved in the transformation process. Falling-ball viscometry and sedimentation analysis of actin solution indicated that cytochalasin cleaved actin filaments and caused depolymerization. The observation of polarized fluorescence of encapsulated actin labeled with acrylodan indicated that the actin filaments in the transformed liposomes aligned along the long axis of the liposomes. Because the actin filaments in the disk- or dumbbell-shaped liposomes formed bundles running along the liposome contour, the transformation was likely to be accompanied by the change in the actin filament arrangement in the liposomes, which was induced by actin-cytochalasin interaction.     

Purification and structural properties of gelsolin, a Ca2+-activated regulatory protein of macrophages

We describe the purification procedure and some of the physiochemical properties of gelsolin, a major Ca2+-dependent regulatory protein of actin gel-sol transformation in rabbit lung macrophages. Gelsolin accounts for the majority of Ca2+ control of actin gelation in macrophage extracts. It is a single polypeptide chain with an average molecular weight of 91,000 a Stokes radius of 44 A, a sedimentation coefficient (s20(0),w) of 4.9 S, an isoelectric point of 6.1, and a frictional ratio of 1.43. Gelsolin binds 2 mol of Ca2+ with high affinity (Ka 1.09 X 10(6) M-1) in the presence of 0.1 M KCl and 2 mM MgCl2.     

Actomyosin-like protein from brain separation and characterization of the actin-like component

1. Actin-like protein (neurin) has been separated from actomyosin-like protein (neurostenin) isolated from bovine brain. This was accomplished by gel filtration chromatography (Sephadex G-200) and by ultracentrifugation in a continuous sucrose gradient containing 0.6 M KI.

2. The actin-like protein stimulated the Mg²⁺-ATPase activity of muscle myosin.

3. It contained bound nucleotide which exchanged with free [¹⁴C]ATP.

4. It polymerized in the presence of 0.1 M KCl and 0.1 mM Mg²⁺ with release of P_i; increase in viscosity occurred upon dilution of the 0.6 M KI to 0.1 M.

5. The neurin reacted immunologically to form a single band with antiserum to neurostenin.

6. The neurin, similar to muscle actin, contained 3-methylhistidine.

7. The sedimentation constant of the protein was 2.8 S.     

Rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex

The rate of capping of actin filaments by the gelsolin-actin complex was measured by inhibition of elongation of the barbed ends of actin filaments. Polymeric actin (0.1-1.0 microM) was added to 0.5 microM monomeric actin and various concentrations of the gelsolin-actin complex (0.08-2.4 nM) to induce nucleated polymerization. As under the experimental conditions (2 mM MgCl2, 100 mM KCl, 37 degrees C, actin monomer concentration less than or equal to 0.5 microM) actin filaments treadmilled, filaments elongated only at the barbed ends and the gelsolin-actin complex did not nucleate actin filaments to polymerize towards the pointed ends. The rate of nucleated actin polymerization in the presence of the gelsolin-actin complex was quantitatively analyzed. The rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex was found to be about 10(7) M-1 s-1.     

The effect of the replacement of ADP with a photoaffinity ATP analogue, 2-azido-ADP, in F-actin on its function

2-Azido-ATP, a photoaffinity ATP analogue, was incorporated into actin and the influence of the incorporation on the actin function was studied. The replacement of ADP with 2-azido-ADP in F-actin both before and after photocross-linking decreased appreciably the actin-activated S1-ATPase activity. Photocross-linked 2-azido-ADP-F-actin could be depolymerized by dialysis against a solution containing 0.1 mM CaCl₂, 0.1 mM ATP and 1 mM Tris-HCl (pH 8.0). However, once it depolymerized, it lost very quickly the ability to polymerize even in the presence of a sufficient amount of ATP and Ca²⁺.     

Interaction of rat liver lysosomal membranes with actin

Membranes were prepared from lysosomes purified 80-fold by centrifugation in a discontinuous metrizamide gradient. When salt- washed membranes were combined with rabbit muscle actin, an increase in viscosity could be measured using a falling ball viscometer. The lysosomal membrane-actin interaction was actin- and membrane- concentration dependent and appeared to be optimal under presumed physiological conditions (2 mM MgCl2, 1 mM MgATP, neutral pH, and free calcium concentration less than 10(-8) M). The actin cross-linking activity of the membrane was optimal at pH 6.4. The interaction was maximal between 10(-7) and 10(-9) M free calcium ions and inhibited by approximately 50% at concentrations of calcium greater than 0.5 x 10(- 7) M. The actin-lysosomal membrane interaction was destroyed if the membranes were pretreated with Pronase, or if the membranes were purified in the absence of protease inhibitors. The interaction was not destroyed if membranes were washed with high salt or extracted with KCl and urea. In addition, a sedimentation assay for the actin-lysosomal membrane interaction was also performed to corroborate the viscometry data. The results suggest the existence of an integral lysosomal membrane actin-binding protein.     

Some properties of Physarum actinin. A regulatory protein of actin polymerization.

A factor termed Physarum actinin was isolated and partially purified from plasmodia of a myxomycete, Physarum polycephalum. When Physarum actinin was mixed with purified Physarum or rabbit striated muscle G-actin in a weight ratio of about 1 actinin to 9 actin and then the polymerization of G-actin induced, G-actin polymerized to the ordinary F-actin on addition of 0.1 M KCl. However, it polymerized to Mg-polymer on addition of 2 mM MgCl2. The reduced viscosity (etasp/C) of the Mg-polymer was 1.2 dl/g, about one-seventh of that of the F-actin (7.4 dl/g). The sedimentation coefficient of the Mg-polymer was 22.8 S, almost the same as that of the F-actin (29.4 S). The Mg-polymer showed the specific ATPase activity of the order of 1 . 10(-3) mumol ATP/mg actin per min. It was shown that Physarum actinin copolymerized with G-actin to form Mg-polymer on addition of 2 mM MgCl2. The molecular weights of Physarum actinin were about 90 000 in salt-free or slat solutions and 43 000 in a dodecyl sulfate solution. The range of salting out with ammonium sulfate was 50--65% saturation, which was different from that of Physarum actin (15--35% saturation). Physarum actinin did not interact with Physarum myosin or muscle heavy meromyosin. When the weight ratio of actinin to actin increased, the flow birefringence of the formed Mg-polymer decreased, and it became almost zero at the weight ratio of 1 actinin to 5 actin. ATPase activity reached the maximum level (2.2 . 10(-3) mumol ATP/mg actin per min) at the same ratio. On the addition of Physarum actinin to purified Physarum F-actin which had been polymerized on addition of 2 mM MgCl2 the viscosity decreased rapidly, suggesting that the F-actin filaments were broken in the smaller fragments or that they transformed to Mg-polymers. A factor with properties similar to Physarum actinin was isolated from acetone powder of sea urchin eggs.     

Aggregation and dispersity of isolated chromaffin granules studied by intensity fluctuation spectroscopy

The aggregation and dispersity of isolated bovine adrenal secretory vesicles (chromaffin granules) were studied by intensity fluctuation spectroscopy. The degree of dispersity and the Z-average translational diffusion coefficients were calculated from the autocorrelation functions of the intensity fluctuations in lase light scattered from the granules in solution. Granules purified by sedimentation through 0.3 M sucrose/Ficoll/²H₂O showed greater dispersity than granules purified by sedimentation through 1.6 M sucrose. By monitoring the scattered light intensity and the diffusion coefficients of the granules, many of the difficulties encountered in the interpretation of absorbance measurements were avoided. Measurements over a range of granule concentrations in sucrose solutions (10 mM HEPES, pH 7.0), indicated that aggregation of the granules occurred at concentrations above 150 μg protein/ml. At low granule concentrations (15–30 μg protein/ml) Ca²⁺-induced aggregation was detected at a threshold of 2–10 mM calcium.     

Properties of two isoforms of human blood platelet alpha-actinin

The structural and functional properties of the aa (2 X 97 kDa) and cc (2 X 94 kDa) isoforms of platelet alpha-actinin have been compared. Structural differences between aa and cc are revealed by their peptide maps, obtained from limited proteolysis, and by their immunological cross-reactivity. Both isoforms stimulate the Mg ATPase activity of actomyosin, bind to F-actin (high-speed sedimentation) and cross-link or gel actin filaments (low-speed sedimentation and viscometry), in a calcium-dependent manner. The study of the interaction with F-actin indicates that the binding of 1 molecule of aa or cc alpha-actinin/9-11 actin monomers is sufficient to produce maximal gelation in the presence of EGTA. CaCl2 at 0.1 mM strongly inhibits the binding of aa to F-actin and weakly that of cc, while it inhibits similarly the cross-linking of either aa or cc. The cross-linking efficiency of cc is 9, 7, 1.7 and 1.3 times higher than that of aa at 4, 20, 30 and 37 degrees C, respectively. The bb form (2 X 96 kDa), which is a proteolytic product of aa [Y. Gache et al. (1984) Biochem. Biophys. Res. Commun. 124, 877-881], behaves roughly as aa, but the calcium sensitivity of its binding to F-actin is intermediate between that of aa and cc. These results suggest that the effect of Ca2+ concentration on the binding of platelet alpha-actinin to F-actin may be partly dissociated from the effect on the cross-linking.     

Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic resonance and sedimentation studies.

The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results.     

Actin‐dependent,Retrograde Motility of Surface‐attached Beads and Aggregating Pigment Granules in Dissociated Teleost Retinal Pigment Epithelial Cells

Teleost retinal pigment epithelial (RPE) cells contain pigment granules within apical projections which undergo actin‐dependent, bi‐directional motility. Dissociated RPE cells in culture attach to the substrate and extend apical projections in a radial array from the central cell body. Pigment granules within projections can be triggered to aggregate or disperse by the presence or absence of 1 mM cAMP. Aminated, fluorescent latex beads attached to the dorsal surface of apical projections and moved in the retrograde direction, towards the cell body. Bead rates on RPE cells with aggregating or fully aggregated pigment granules were 2.2 ± 0.5 and 2.6 ± 0.2 μm/min (mean ± SEM), respectively, similar to rates of aggregating (retrograde) pigment granule movement (2.0 ± 0.4 μm/min). Bead rates were slightly slower on cells with fully dispersed or dispersing pigment granules (1.5 ± 0.1 and 1.5 ± 0.4 μm/min). Movements of surface‐attached beads and aggregating pigment granules were closely correlated in the distal portions of apical projections, but were more independent of each other in proximal regions of the projections. The actin disrupting drug, cytochalasin D (CD), reversibly halted retrograde bead movements, suggesting that motility of surface‐attached particles is actin‐dependent. In contrast, the microtubule depolymerizing drug, nocodazole, had no effect on retrograde bead motility. The similar characteristics and actin‐dependence of retrograde bead movements and aggregating pigment granules suggest a correlation between these two processes.     

Calcium-insensitive binding of heavy meromyosin to regulated actin at physiological ionic strength

Several conflicting reports have been made regarding the affinity of myosin heads (subfragment 1 and heavy meromyosin (HMM) for regulated actin (actin complexed with tropomyosin and troponin) at low ionic strength (mu = 18-50 mM) and whether or not this interaction is Ca2+ sensitive (Chalovich, J. M., and Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437; Chalovich, J. M., and Eisenberg, E. (1984) Biophys. J. 45, 221a; Wagner, P. D., and Stone, D. B. (1983) Biochemistry 22, 1334-1342; and Wagner, P. D. (1984) Biochemistry 23, 5950-5956). Since the low ionic strengths used in the above studies do not represent the physiological ionic strength under which intact muscle exhibits Ca2+-dependent tension development, we investigated the possibility of whether a Ca2+-dependent regulated actin-HMM interaction could be observed at physiological ionic strength (mu = 134 mM, pH 7.4) and in the presence of ATP (at 23-24 degrees C). Direct binding of HMM to varied concentrations of regulated actin (87.7-221 microM free actin) was measured by sedimentation in an air-driven ultracentrifuge. Under the above conditions, we found that the regulated actin activation of HMM-Mg2+-ATPase was about 94% inhibited in the absence of Ca2+ although the association constant (Ka) is only moderately affected in the presence of Ca2+. These results are similar to those obtained by Chalovich and Eisenberg (1982 and 1984) with subfragment 1 and HMM, respectively, at low ionic strength and support their suggestion that in solution tropomyosin-troponin may not act totally by physically blocking the formation of cross-bridges with actin, but instead may act to inhibit a kinetic step in the overall ATPase rate. Whether this holds true in more intact systems (e.g. myosin, thick filaments) remains to be determined. Our results also show a good correlation between levels of ATPase activation and HMM binding by unregulated actin and in regulated actin in the presence of Ca2+.