The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts

The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemmaectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.     

The molecular mobility of alpha-actinin and actin in a reconstituted model of gelation

Dictyostelium discoideum alpha-actinin (D.d. alpha-actinin) is a calcium and pH-regulated actin-binding protein that can cross-link F-actin into a gel at a submicromolar free calcium concentration and a pH less than 7 [Fechheimer, et al., 1982]. We examined mixtures of actin and D.d. alpha-actinin at four pH and calcium concentrations that exhibited various degrees of gelation or solation. The macroscopic viscosities of these mixtures were measured by falling ball viscometry (FBV) and compared to the translational diffusion coefficients measured by gaussian spot and periodic-pattern fluorescence photobleaching recovery (FPR) of both the actin filaments and D.d. alpha-actinin. A homogeneous, macroscopic gel was not composed of a static actin network. Instead, the filament diffusion coefficient decreased to approximately 65% of the control value. If the D.d. alpha-actinin concentration was increased, the solution became inhomogeneous, consisting of domains of higher actin concentration. These domains were often composed of a static actin network. The mobility of D.d. alpha-actinin consisted of a major fraction that freely diffused and a minor fraction that appeared immobile under the conditions employed. This suggested that D.d. alpha-actinin binding to the actin filaments was static over the time course of measurement (approximately 5 sec). Under solation conditions, there was no apparent interaction of actin with D.d. alpha-actinin. These results demonstrate that 1) actin filaments need not be cross-linked into an immobile, static array in order to have macroscopic properties of a gel; 2) interpretation of the rheological properties of actin:alpha-actinin gels are complicated by spatial heterogeneity of the filament concentration and mobility; and 3) a fraction of D.d. alpha-actinin binds statically to actin in undisturbed gels. The implications of these results are discussed in relation to cytoplasmic structure and contractility.     

Contraction of Dictyostelium ghosts reconstituted with myosin II.

Cytoskeletons, or 'Triton ghosts,' that contained mainly actin and myosin II were prepared from Dictyostelium discoideum amoebae by extraction with Triton X-100. The Triton ghosts contracted immediately upon addition of ATP. However, under high-salt conditions in the presence of ATP, they did not contract but released myosin II. Almost all of the applied myosin II became associated with ghosts when myosin-free Triton ghosts, prepared in this way, were incubated with purified actin and then with myosin II from Dictyostelium. Immunofluorescence microscopy demonstrated that the associated myosin was localized in the cortical actin layer of the ghosts. Furthermore, the ghosts reconstituted with purified myosin resumed ATP-dependent contraction. Skeletal muscle myosin could also restore contractility to ghosts from which myosin had been extracted. The amount of myosin II necessary for the contraction of the ghosts was calculated by two methods. Less than 10% of the myosin II in intact cells was necessary for the contraction. These results show that myosin II is responsible for the contraction of the Dictyostelium cytoskeleton.     

Isolation and characterization of a 30,000-dalton calcium-sensitive actin cross-linking protein from Dictyostelium discoideum

A calcium-sensitive actin-binding protein having a subunit molecular mass of 30,000 daltons (30K protein) has been isolated from Dictyostelium discoideum. Structural, immunological, and functional analyses demonstrated that the 30K protein was distinct from other actin-binding proteins of D. discoideum. A native molecular mass of 31,700 daltons was determined by equilibrium sedimentation, indicating that the protein is monomeric. The Stokes radius was 30 A. The frictional coefficient calculated from these measurements was 1.44, indicating an asymmetric shape. The 30K protein induced an increase in the viscosity of a solution of F-actin. Bundles of actin filaments were observed in negatively stained mixtures of actin and the 30K protein. Both the formation of filament bundles and the increases in viscosity of actin induced by the 30K protein were observed in the presence of 1 X 10(-8) M but not 2 X 10(-6) M calcium. Variation of the pH from 6.6 to 7.8 had no effect on the activity of the 30K protein. Calcium induced neither a large change in quaternary structure of the 30K protein nor a restriction of the lengths of actin filaments by the 30K protein. The apparent affinity of the 30K protein for actin was decreased in the presence of calcium. Reversible cross-linking of actin filaments by the 30K protein may contribute to regulation of the consistency and contractility of cytoplasm in D. discoideum.     

THE CONTRACTILE BASIS OF AMOEBOID MOVEMENT : I. The Chemical Control of Motility in Isolated Cytoplasm

Cytoplasm has been isolated from single amoeba (Chaos carolinensis) in physiological solutions similar to rigor, contraction, and relaxation solutions designed to control the contractile state of vertebrate striated muscle. Contractions of the isolated cytoplasm are elicited by free calcium ion concentrations above ca. 7.0 x 10^-7 M. Amoeba cytoplasmic contractility has been cycled repeatedly through stabilized (rigor), contracted, and relaxed states by manipulating the exogenous free calcium and ATP concentrations. The transition from stabilized state to relaxed state was characterized by a loss of viscoelasticity which was monitored as changes in the capacity of the cytoplasm to exhibit strain birefringence when stretched. When the stabilized cytoplasm was stretched, birefringent fibrils were observed. Thin sections of those fibrils showed thick (150–250 Å) and thin (70 Å) filaments aligned parallel to the long axis of fibrils visible with the light microscope. Negatively stained cytoplasm treated with relaxation solution showed dissociated thick and thin filaments morphologically identical with myosin aggregates and purified actin, respectively, from vertebrate striated muscle. In the presence of threshold buffered free calcium, ATP, and magnesium ions, controlled localized contractions caused membrane-less pseudopodia to extend into the solution from the cytoplasmic mass. These experiments shed new light on the contractile basis of cytoplasmic streaming and pseudopod extension, the chemical control of contractility in the amoeba cytoplasm, the site of application of the motive force for amoeboid movement, and the nature of the rheological transformations associated with the circulation of cytoplasm in intact amoeba.     

Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking

Gelsolin is a calcium binding protein that shortens actin filaments. This effect occurs in the presence but not in the absence of micromolar calcium ion concentrations and is partially reversed following removal of calcium ions. Once two actin molecules have bound to gelsolin in solutions containing Ca2+, one of the actins remains bound following chelation of calcium, so that the reversal of gelsolin's effect cannot be accounted for simply by its dissociation from the ends of the shortened filaments to allow for elongation. In this paper, the interactions with actin of the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stable 1:1 gelsolin-actin complexes are compared with those of free gelsolin. The abilities of free or complexed gelsolin to sever actin filaments, nucleate filament assembly, bind to the fast growing (+) filament ends, and lower the filament size distribution in the presence of either Ca2+ or EGTA were examined. The results show that both free gelsolin and gelsolin-actin complexes are highly dependent on Ca2+ concentration when present in a molar ratio to actin less than 1:50. The gelsolin-actin complexes, however, differ from free gelsolin in that they have a higher affinity for (+) filament ends in EGTA and they cannot sever filaments in calcium. The limited reversal of actin-gelsolin binding following removal of calcium and the calcium sensitivity of nucleation by complexes suggest an alternative to reannealing of shortened filaments that involves redistribution of actin monomers and may account for the calcium-sensitive functional reversibility of the solation of actin by gelsolin.     

Calcium-sensitive binding of heavy meromyosin to regulated actin requires light chain 2 and the head-tail junction

Sedimentation in a preparative ultracentrifuge was used to determine the affinity of heavy meromyosin, HMM, for regulated actin, F-actin plus troponin-tropomyosin, in the presence of MgATP at pH 7.0, 20 degrees C, and mu = 18 mM. HMM was prepared from vertebrate striated muscle myosin by a mild chymotryptic digestion. This HMM contained 85-90% intact 19 000-dalton light chains, LC2. In the presence of calcium, 90% of the HMM bound to regulated actin with an association constant of (2-4) X 10(4) M-1. In the absence of calcium, while one-third of the HMM bound with an affinity similar to that observed in the presence of calcium, the rest bound much more weakly. It was not possible to accurately determine the association constant for this weakly binding HMM, but a 20-fold reduction in affinity is consistent with the binding data. The binding of single-headed heavy meromyosin to regulated actin was similarly sensitive to the calcium concentration. Since removal of calcium inhibits the regulated actin-activated ATPase of HMM greater than 20-fold, troponin-tropomyosin must be capable of inhibiting both the binding of HMM to regulated actin and a step which occurs after binding but prior to product release. Removal of LC2 increased the fraction of HMM with calcium-insensitive binding, and adding LC2 back to this depleted HMM restored most of the calcium sensitivity. Chymotryptic cleavage of LC2 to a 17 000-dalton fragment destroyed the calcium-sensitive binding of HMM to regulated actin. Phosphorylation of LC2, however, had no detectable effect on this binding. Thus, the calcium-sensitive binding of HMM to regulated actin requires that both the head-tail junction and the N-terminal part of LC2 be intact. Binding studies with cross-linked regulated actins and kinetic measurements of the rates of change in turbidity demonstrate that this calcium sensitivity is due to calcium binding to troponin and not to LC2.     

Abundance, relative gelation activity, and distribution of the 95,000- dalton actin-binding protein from Dictyostelium discoideum

We have studied the abundance, relative gelation activity, and distribution of the 95,000-dalton actin-binding protein in Dictyostelium discoideum amoebae. The 95,000-dalton protein was a prominent polypeptide as assessed using quantitative densitometry and radioimmunoassay. We estimated that this protein comprised approximately 1.2% of the protein in a soluble extract of amoebae. The molar ratio of the dimeric 95,000-dalton protein to actin in the soluble extract was 1:30. The apparent viscosities of actin mixtures with either the purified 95,000-dalton protein or the soluble extract were measured by falling ball viscometry in an attempt to assess the contribution of the 95,000-dalton protein to gelation of the soluble extract. The gelation of the soluble extract was significantly less than that expected from the contribution of the 95,000-dalton protein alone. Consequently, we questioned the validity of quantitative analyses of the contributions of specific actin-binding proteins to the gelation of cell extracts. The apparent distribution of the 95,000- dalton protein was observed in chemically fixed and extracted cells by immunofluorescence microscopy and compared with the distribution of cytoplasm and organelles visible using light microscopy. The 95,000- dalton protein was dispersed throughout the cytoplasm of fixed cells, was apparently excluded from prominent organelles, and displayed brightest fluorescence in regions of hyaline cytoplasm. These regions of hyaline cytoplasm that exhibited the brightest fluorescence were observed in the cortical region of rounded cells and in pseudopods of polarized cells. Thus, cell shape and polarity may also have influenced the apparent distribution of the 95,000-dalton protein observed by immunofluorescence microscopy. Study of the distribution of fluorescein- labeled ovalbumin injected into living cells supported the interpretation that the thickness of the cell and the distribution of organelles contributed to the apparent distribution of the 95,000- dalton protein observed in fixed cells using immunofluorescence microscopy. We suggest that the 95,000-dalton protein contributes to modulation of the consistency and contractility of the cytoplasm of D. discoideum amoebae, since it could cross-link actin filaments in vitro in a reversible process that was regulated by changes in the concentration of calcium and of protons, and since it was present in large quantity in the cytoplasm of these cells.     

Dephosphorylation of myosin by the catalytic subunit of a type-2 phosphatase produces relaxation of chemically skinned uterine smooth muscle

It is now well-established that phosphorylation of the 20,000-dalton light chain of smooth muscle myosin (LC20) is a prerequisite for muscle contraction. However, the relationship between myosin dephosphorylation and muscle relaxation remains controversial. In the present study, we utilized a highly purified catalytic subunit of a type-2, skeletal muscle phosphoprotein phosphatase (protein phosphatase 2A) and a glycerinated smooth muscle preparation to determine if myosin dephosphorylation, in the presence of saturating calcium and calmodulin, would cause relaxation of contracted uterine smooth muscle. Addition of the phosphatase catalytic subunit (0.28 microM) to the muscle bath produced complete relaxation of the muscle. The phosphatase-induced relaxation could be reversed by adding to the muscle bath either purified, thiophosphorylated, chicken gizzard 20,000-dalton myosin light chains or purified, chicken gizzard myosin light chain kinase. Incubation of skinned muscles with adenosine 5'-O-(thiotriphosphate) prior to the addition of phosphatase resulted in the incorporation of 0.93 mol of PO4/mol of LC20 and prevented phosphatase-induced relaxation. Under all of the above conditions, changes in steady-state isometric force were associated with parallel changes in myosin light chain phosphorylation over a range of phosphorylation extending from 0.01 to 0.97 mol of PO4/mol of LC20. We found no evidence that dephosphorylation of contracted uterine smooth muscles, in the presence of calcium and calmodulin, could produce a latch-state where isometric force was maintained in the absence of myosin light chain phosphorylation. These results show that phosphorylation or dephosphorylation of the 20,000-dalton myosin light chain is adequate for the regulation of contraction or relaxation, respectively, in glycerinated uterine smooth muscle.     

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.     

A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner

A 40,000-dalton protein that affects the assembly properties of actin in a Ca2+-dependent manner has been purified from Dictyostelium discoideum. Gel filtration chromatography indicates that the native form of this protein is a monomer. A major effect of this protein is to reduce the sedimentability of F-actin in a stoichiometric fashion. Nearly complete loss of sedimentability is observed at ratios of the 40,000-dalton protein to actin of greater than 1:10. At low stoichiometries, this protein can accelerate the rate of actin assembly under certain experimental conditions. These effects of the 40,000- dalton protein on the actin assembly properties described above require calcium ion. The 40,000-dalton protein does not exert its effects by proteolyzing actin. Furthermore, peptide maps demonstrate that this protein is not a proteolytic fragment of actin.     

The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells

Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl, and a reducing agent. Gelation was inhibited reversibly by 0.5 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.     

A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments

We have purified an actin binding protein from amebas of Dictyostelium discoideum which we call 95,000-dalton protein (95K). This protein is rod shaped, approximately 40 nm long in the electron microscope, contains two subunits measuring 95,000 daltons each, and cross-links actin filaments. Cross-linking activity was demonstrated by using falling-ball viscometry, Ostwald viscometry, and electron microscopy. Cross-linking activity is optimal at 0.1 microM Ca++ and pH 6.8, but is progressively inhibited at higher Ca++ and pH levels over a physiological range. Half-maximal inhibition occurs at 1.6 microM free Ca++ and pH 7.3, respectively. Sedimentation experiments demonstrate that elevated Ca++ and pH inhibit the binding of 95K to F-actin which explains the loss of cross-linking activity. Electron microscopy demonstrates that under optimal conditions for cross-linking, 95K protein bundles actin filaments and that this bundling is inhibited by microM Ca++. Severing of actin filaments by 95K was not observed in any of the various assays under any of the solution conditions used. Hence, 95K protein is a rod-shaped, dimeric, Ca++- and pH-regulated actin binding protein that cross-links but does not sever actin filaments.     

In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells

We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contractions of the tail cortex in many amoeboid cells as defined by the solation-contraction coupling hypothesis (Taylor, D. L., and M. Fechheimer. 1982. Phil. Trans. Soc. Lond. B. 299:185-197). The competing processes of solation and contraction of the gel would appear to be mutually exclusive. However, it is the temporal-spatial balance of the rate and extent of two stages of solation, coupled to contraction, that can explain the conversion of gelled ectoplasm in the tail to a solated endoplasm within the same small volume, generation of a force for the retraction of tails, maintenance of cell polarity, and creation of a positive hydrostatic pressure to push against the newly formed endoplasm. The mechanism of solation-contraction of cortical cytoplasm may be a general component of the normal movement of a variety of amoeboid cells and may also be a component of other contractile events such as cytokinesis.     

The Contractile Proteins and Calcium Mediated Gelation and Contraction of Pollen Tube Extracts

The crude extracts of pollen tubes, like other nonmuscle ceils, showed gelation at Iow Ga2+ concentrations and ATP-dependent contraction at higher Ga2+ concentrations. The contracted cytoplasmic clots contained a lot of filaments which were mainly composed of actin, myosin, 105 kD, 67 kD, 48 kD, 38 kD, 34 kD and 28 kD proteins. It is likely that Ca2+ are able to mediate tranformation of acfin from a less ordered state to a more oriented filaments, which interact with actin-binding proteins to form the filamentous network, thus to induce the gel formation of cytoplasm, to regulate the interaction of actin and myosin which transform the chemical energy of ATP into mechanical work of contractile movement of cytoplasm.     

A calcium-sensitive preparation from Physarum polycephalum.

Differential ultracentrifugation of an extract of the plasmodium of Physarum polycephalum yields a high-speed fraction which exhibits calcium-sensitive adenosine triphosphate activity at low ionic strength. The rate of inorganic phosphate production increased from 2- to 25-fold in different preparations when the calcium concentration was increased from about 10(-8) to 10(-5) M. Complement fixation using specific antibody to Physarum myosin showed the fraction to contain 3% myosin. By electron microscopy, actin-like microfilaments 50--150 nm long were present. Addition of pure rabbit F-actin or myosin to this fraction activated the ATPase measured in EGTA and so partially reversed the calcium sensitivity. If muscle myosin was added to the supernatant from which the fraction was centrifuged, a "hybrid complex" was obtained which included actin and additional protein from the plasmodium, and this hybrid was also calcium sensitive. Over 85% of the calcium-sensitive, magnesium-activated ATPase could be precipitated by sequential "hybrid" formation. The calcium sensitivity of the hybrid was maximal when formed at the lowest ratios of added myosin to Physarum proteins. It is concluded that the results do not allow a simple interpretation along the lines of either actin-linked or myosin-linked sensitivity. Evidence consistent with both a form of actin-linked and myosin-linked sensitivity is present in our results.     

Reactivation of contraction in detergent-lysed teleost retinal cones

Teleost retinal cones contract in the light and elongate in the dark. In the green sunfish, Lepomis cyanellus, the necklike myoid region of the cone contracts from as much as 120 micrometers (midnight dark- adapted) to 6 micrometers in fully light-adapted state. When dark- adapted fish are exposed to light (1.4 lux), cone myoids contract with a linear rate of 1.5 +/- 0.1 micrometers/min. We report here that detergent-lysed motile models of teleost retinal cones exhibit calcium- and ATP-dependent reactivated contraction, with morphology and rate comparable to that observed in vivo. For reactivation studies isolated dark-adapted retinas were lysed with nonionic detergent Brij-58 (0.1- 1.0%). In reactivation medium containing 10(-5) M free calcium and 4 mM ATP, the lysed cones contracted with normal morphology at in vivo rates (1.4 +/- 1 micrometer/min). Little contraction was observed if ATP or detergent was deleted from the medium or if free calcium levels were less than 10(-8) M. Ultrastructural examination of cone models lysed with 1% Brij-58 revealed that, in spite of extensive extraction of the cytoplasmic matrix, cytoskeletal components (thin filaments, intermediate filaments, microtubules) were still present. Thus we have produced extensively extracted motile models of teleost retinal cones which undergo calcium- and ATP-dependent reactivated contraction with normal morphology at physiological rate.     

The Ca2+ sensitivity of liver gelactin

The Ca2+ sensitivity of liver gelactin-induced actin gelation was reinvestigated by low-shear viscosity using the falling-ball technique. By this technique, we demonstrate that the gelatin of actin by gelactin can be influenced by the presence of calcium ions depending on the concentrations of both proteins, actin and gelactin. At low concentrations of gelactin, the gelatin of actin exhibits a bell-shaped dependency on free calcium ion concentration, being stimulated between pCa 8 and 6 and inhibited at pCa below 5.5, while at high gelactin concentrations the calcium sensitivity of actin gelation is apparently abolished. Although the sensitivity observed in the physiological range of calcium concentrations may be of importance in vivo, the sensitivity observed at higher calcium concentrations more probably reflects the state of actin polymerization in different ionic conditions. These results confirm our previous conclusions on the peculiarity of gelactin as an F-actin cross-linker.     

Purification of calcium-sensitive regulatory protein of platelets which inhibits the gelation of actin

Ca²⁺-sensitive regulatory protein of human platelets which inhibits the gelation of actin was purified by DEAE-Sepharose and an affinity column using actin as a ligand. The protein was a single polypeptide chain with an average molecular weight of 90,000 and it bound to actin and inhibited its gelation at concentration from 10^?6–10^?7M of free calcium. Since the protein existed in the form of a complex with actin even though at concentration lower than 10^?7M of free calcium, binding and dissociation of actin and the protein appeared to be dependent on the concentration of free calcium, and complete dissociation was not seen.