Avraham Oplatka

Destabilization of actin filaments as a requirement for the secretion of catecholamines from permeabilized chromaffin cells

In the search for a functional role of cytoskeletal proteins in the mechanism(s) of stimulus‐secretion coupling, we have previously demonstrated that the actomyosin system might be involved in the transport of cations across the plasma membrane of bovine adrenal chromaffin cells [(1986) J. Biol. Chem. 261, 5745‐5750]. To establish whether actin and myosin might also be involved in later stages of the cellular response, we have examined the possible effects of various actin‐specific reagents on the calcium‐mediated secretion of catecholamines from digitonin‐permeabilized cells. F‐Actin‐destabilizing agents, such as cytochalasin D or DNase 1, were found to promote Ca2+‐stimulated (as well as basal) secretion. By contrast, stabilizers, like phalloidin, produced the opposite effect. It is concluded that stimulus‐secretion coupling in chromaffin cells might require the reorganization of actin for modulating both ion transport across the plasma membrane and exocytotic secretion per se.

Enzymatic mechanochemistry. I. The interaction of heavy meromyosin with “immobilized adenosine triphosphate”

Abstract ATP was covalently bound to an agarose gel. The insolubilized ATP was found to be capable of specifically binding heavy meromyosin. The adsorbed heavy meromyosin could be eluted by ATP in solution. Both binding and elution by ATP of heavy meromyosin were not much effected by Ca 2+ , Mg 2+ or EDTA. While the water-soluble polyalanine-myosin was also found to be adsorbed, myosin in 0.5 M KCl did not seem to be adsorbed by agarose-ATP. Both Mg 2+ and Ca 2+ appear to activate the splitting of bound ATP by heavy meromyosin to practically the same extent. We prepared water-soluble derivatives of ATP in which ATP underwent the same chemical modification required for its coupling to agarose but in which the agarose component was absent. Their splitting by heavy meromyosin was also activated by Mg 2+ though to a lesser extent but actin did not influence this reaction. Possible relations between our findings and the various stages of the reaction between myosin and ATP, as well as the potential use of columns filled with insolubilized NTPs for the separation and purification of myosin and of its subfragments, are discussed.

Physico-chemical studies on the light chains of myosin III. Evidence for a regulatory role of a rabbit myosin light chain☆

Abstract Treatment of myosin with DTNB causes a decrease in the calcium sensitivity of actomyosin, concurrently with the release of the DTNB light chains. The removal of the calcium-binding DTNB light chains is accompanied by a loss of the calcium binding capacity of myosin. A regulatory role is ascribed to these light chains.

Fibrin-Blood Platelet Interaction in a Contracting Clot

The interaction of fibrin and blood platelets was studied by measuring tension development in clots containing different ratios of these two components. Isometric tension attained a saturating value (MT) when plotted as a function of the concentration of either component at a constant concentration of the other. The value of MT increases linearly with the minimal platelet concentration (Ps) required in order to reach saturation at various different fibrin concentrations. A linear relationship also holds in a plot of Ps versus the logarithm of the corresponding fibrin concentration. When the cylindrical clots contracted isotonically, the velocity, normalized with respect to the momentary length, was found to be constant. This velocity decreased in an hyperbolic manner with increasing fibrin concentration at a constant concentration of blood platelets. The results are interpreted in terms of a three‐dimensional network in which the blood platelets serve as contractile cross‐bridges connecting fibrin fibres. Tension development is assumed to be accompanied by the reduction of the angle between fibrin fibres. An attempt was made to calculate the isometric tension developed by a platelet.

The possible implication of membrane-associated actin in stimulus-secretion coupling in adrenal chromaffin cells

Chromaffin cells of the bovine adrenal medulla were fused with liposomes containing DNAaseI. Resting membrane potential measurements, obtained by the use of the cyanine dye diS-C3 (5), showed that DNAaseI incorporation causes depolarization from −56 to −31 mV, which is similar to that induced by ouabain. The level of basal secretion which occurs after the introduction of DNAaseI is increased several fold. Actin filaments, which can be depolymerized by DNAaseI, thus appear to be involved in stimulus-secretion coupling in chromaffin cells. It is suggested that plasma membrane-associated microfilaments control the Na+-K+-pump and/or the permeability of Ca2+ ions thus affecting the membrane potential as well as secretion.

Tension development in skinned glycerinated rabbit psoas fiber segments irrigated with soluble myosin fragments

Single glycerinated rabbit psoas muscle fibers were skinned by splitting them lengthwise. The fiber segments thus obtained were more easily accessible to solutes in the surrounding medium than the intact fibers. Using such segments, active tension could be fully abolished by adding N-ethylmaleimide under conditions which lead to inhibition of actin activation of the ATPase activity of myosin. Such muscles could, however, develop tension after irrigation with myosin or with the water-soluble active myosin fragments heavy meromyosin (HMM) or its subfragment 1 (HMM-S1). The induced tensions increased with increasing protein concentration in the irrigating solution. At any given protein concentration, the tension generated by myosin was larger than that produced by HMM which was, in turn, greater than that induced by HMM-S1 e.g. at 15 mg/ml protein the tensions produced by these three myosin moieties were 44.0, 14.0 and 2.8 g/cm2, respectively. The tension was found to be intimately associated with ATP splitting; thus, HMM and HMM-S1 which have been treated with reagents abolishing actin-activated ATPase failed to induce tension development. A contractile force may thus be generated through the interaction with actin of the water-soluble, enzymatically active, myosin subfragments involving the splitting of ATP.

Active streaming in actomyosin solutions.

Abstract Solutions of actomyosin, extracted from plasmodia of Physarum polycephalum or from rabbits striated muscle, were introduced into microcapillaries in the presence of ATP. Vigorous and sometimes oscillatory streaming was observed which resembled in many respects cytoplasmatic streaming.

Phosphatidylserine directs differential phosphorylation of actin and glyceraldehyde-3-phosphate dehydrogenase by protein kinase C: possible implications for regulation of actin polymerization.

The phospholipid‐dependent protein kinase C is implicated in the regulation of cellular motility and energy metabolism. Phosphatidylserine, a main cofactor of protein kinase C, is involved in the regulation of glyceraldhehyde‐3‐phosphate dehydrogenase, which as actin, was shown to be phosphorylated by purified protein kinase C. Here, we study the effect of phosphatidylserine on the enzyme‐substrate interaction of protein kinase C with glyceraldhehyde‐3‐phosphate dehydrogenase and actin. The stoichiometry of glyceraldhehyde‐3‐phosphate dehydrogenase phosphorylation is not affected by varying the level of phosphatidylserine. However, actin phosphorylation is dependent on phosphatidylserine level, peaking at high phosphatidylserine concentration. Moreover, if actin and glyceraldhehyde‐3‐phosphate dehydrogenase are cophosphorylated at high phosphatidylserine concentration, actin phosphorylation is favored, despite lower affinity for protein kinase C. Hence, phosphatidylserine directs differential phosphorylation of these key proteins of glycolysis and cellular motility and might be capable of recruiting protein kinase C for preferential actin phosphorylation. The sedimentation of phosphorylated actin is increased 3.8 fold and total actin 1.7 fold, suggesting that phosphorylation promotes actin polymerization.

Rabbit skeletal muscle F-actin can be stable at low ionic strength, provided trace amounts of Ca2+ are absent

Addition of low concentrations (0.2--2.0 mM) of EGTA to rabbit skeletal muscle G-actin in the presence of ATP caused increase in viscosity. The effect is probably due to chelation of Ca2+. EGTA-polymerized actin was sedimented in the ultracentrifuge as a pellet which could be depolymerized in the presence of Ca2+ and then repolymerized. Electron microscopy indicated that formation of filamentous actin which appears to be somewhat more flexible than F-actin obtained by polymerization with KCl. The EGTA-polymerized actin was dissociated by DNAase I faster than KCl-polymerized actin. F-Actin can thus be stable also in very low ionic strength media if Ca2+ is removed whereas for G-actin to be the only form of the protein in such media, micromolar concentrations of Ca2+ must be present.

PHOSPHORYLATION OF FIBROBLAST MYOSIN

1. Introduction The fact that myosin might be phosphorylated has been known for more than a decade [l] . However, only recently have special kinases been described, in striated muscle [2] and in platelets [3] , which catalyze the phosphorylation of one of the light-chains of myosin. The myosin light-chain kinase isolated from platelets was found to be capable of transferring the terminal-phosphate of y-labeled AT”P to the 20 000 dalton light-chain of platelet myosin and to the same light-chain in smooth-muscle, flbroblast and rhabdomyosarcoma myosin but it does not affect striated muscle myosin [4] . It was suggested that this phenomenon may have a physiological significance as the actin-activated ATPase activity of platelet myosin increases after phosphorylation [.5]. Considering the possibility that the phosphorylation of myosin may play a role in the regulation of cell mobility it was interesting to look for endogenous myosin phosphorylat- ing activity in other non-muscle cells such as fibro- blasts. So far there is only one indication (in the case of cardiac myosin [6,7]) that the heavy-chain of myosin, in addition to 20 000 and 18 500 dalton light-chains, might be phosphorylated. In the following we will show that both heavy and light chains oi fibroblast myosin can be phosphorylated by incubating fibro- blast extracts with ATP.