Reconstituted Actomyosin Research Articles

Differential Actin Binding Affinity Leads to Protein Sorting in a Reconstituted Active Composite Layer

Various functional cell surface proteins bind to cortical actin and undergo transient clustering driven by actomyosin flows. Examples include the GPI-anchored proteins, Ras-signalling proteins, T-cell receptors and many glycoproteins such as CD44. Given the density and diversity of protein and lipid species in the plasma membrane, the question arises how the local accumulation of specific molecules is achieved in a timely manner. Here we explore the possibility that the combination of differential binding of surface molecules to actin and the non-equilibrium acto-myosin dynamics leads to molecular patterning and cluster formation of membrane proteins. Using reconstituted acto-myosin networks tethered to supported lipid bilayers, as well as theoretical simulations, we provide evidence for this proposed active sorting mechanism. We show that differential affinity for actin templates a variety of patterns of membrane tethered proteins, including complete segregation, bull's eye and mixed pattern. Our results suggest that this mechanism could help in understanding the spatio-temporal organization and regulation of various processes at the cell surface.

Thermal gelation profile changes in reconstituted actomyosin due to storage under a high salt concentration and low temperature.

Changes in the heat-induced gelation properties of reconstituted rabbit skeletal actomyosin stored under a high salt concentration at pH 6.0 and 0 degree C were investigated at different weight ratios of actin to myosin by using dynamic rheological and biochemical measurements. The addition of actin resulted in a pronounced peak maximum at about 50 degrees C and an accompanying temporary reduction in the range at about 50 degrees C to 60 degrees C. The more the initial actin concentration was increased, the greater was the area of the peak/shoulder. However, this area was markedly diminished with increasing storage time. As a result, the dynamic rheological pattern was transformed from an actomyosin type into a myosin type. The relationship between the G' value at 80 degrees C and the actin/myosin weight ratio was curvilinear, with a peak at the ratio of 0.05, immediately after storage was started. This profile changed during storage, depending on the extent to denaturation of actin and myosin in the reconstituted actomyosin (RAM). The G' value of actomyosin in 0.5 M KCl with a small actin/myosin ratio of 0.05 decreased to one-half of its initial value after 7 days of storage, whereas the G' value with a large actin/myosin ratio of 0.225 increased by about 1.6 times. In 1.5 M KCl, all the G' values declined to the level with myosin alone after 7 days of storage. The time-course plots of the remaining actin concentration in RAM at different weight ratios of actin to myosin after being treated with 0.5 M or 1.5 M KCl showed a decrease in the actin content with increasing storage time, and an increase in the KCl concentration to 1.5 M KCl promoted the denaturation of actin in RAM faster than with 0.5 M KCl. The surface hydrophobicity of each RAM sample progressively increased with increasing storage time, while little significant increase in the sulfhydryl (SH) content during storage was observed. It is concluded that changes in the heat-induced gelation properties of actomyosin during storage are largely attributable to the denaturation of actin rather than to the denaturation of myosin or to quantitative changes in the SH content and hydrophobicity.

Amino Acid Sequence of C-Terminal 17 kDa CNBr-Fragment of Akazara Scallop Troponin-I1

The M(r) 52,000 subunit of Akazara scallop striated muscle troponin, which was tentatively identified as troponin I, was cleaved into two major fragments with CNBr: C-terminal 17 kDa fragment (CN17K) and N-terminal 35 kDa fragment (CN35K) [J. Biochem. 108, 519-521 (1990)]. CN17K inhibits rabbit reconstituted actomyosin Mg-ATPase activity, weakly in the absence of troponin T but strongly in its presence, together with Akazara tropomyosin. CN35K, however, hardly shows such inhibition. Thus, the amino acid sequence of the CN17K was determined by the Edman method. CN17K comprises 135 amino acid residues and its calculated molecular mass is 15,732 Da. A computer search of the SWISS-PROT data base revealed the TnIs of crayfish tail muscle, rabbit skeletal muscle, and bovine cardiac muscle to be homologous proteins with total sequence homologies of 39, 30, and 30%, respectively, to CN17K. Significantly high homology was observed among these TnIs in the regions around residues 75-95, 99-114, and 135-151 of the rabbit TnI. From these facts, we conclude that the 52K subunit is a TnI.

Comparative studies on biochemical characteristics of troponins from ezo-giant scallop ( patinopecten yessoensis) and akazara scallop ( chlamys nipponesis akazara)

1. 1. Tropinins were isolated from the striated adductor muscle of ezo-giant scallop and akazara scallop, and biochemical properties were comparatively studied. 2. 2. The ezo-giant scallop and akazara scallop troponins conferred the same extent of Ca 2+-sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, and the Ca 2+ concentration required for a half-maximal activation, with either troponin, was estimated as 1 μM. 3. 3. The ezo-giant scallop troponin consisted of three subunits of M r , 52,000, 40,000 and 20,000, and they were regarded as troponin-I, -T and -C, respectively, from the hybridization test with the akazara scallop troponin subunits which we previously identified [Ojima and Nashita (1988) J. Biochem. 104, 207–210].

A Binary Complex of Troponin-I and Troponin-T from Akazara Scallop Striated Adductor Muscle1

A binary complex consisting of Mr 19,000 and Mr 40,000 components was co-purified with troponin from a crude troponin fraction of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle. This complex is incapable of conferring Ca(2+)-sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, rather strongly inhibiting it, but became capable on further complexing with Akazara scallop troponin-C. To examine the effects of the Mr 19,000 and Mr 40,000 components on the ATPase activity, they were separated from each other by CM-Toyopearl column chromatography. The Mr 19,000 component strongly inhibited the Mg-ATPase activity of actomyosin-tropomyosin and the inhibition was reversed by further addition of the Akazara scallop troponin-C. On the other hand, the Mr 40,000 component slightly increased it. On hybridization with the Akazara scallop troponin subunits, the Mr 19,000 and Mr 40,000 components were shown to be able to substitute for troponin-I and troponin-T, respectively. The amino acid compositions of the Mr 40,000 component and troponin-T were almost identical, and those of the Mr 19,000 component and Mr 17,000 C-terminal fragment of the troponin-I resembled each other fairly well. From these results, it may be concluded that the Mr 19,000-40,000 binary complex is the troponin-I-troponin-T complex.

American Lobster Troponin1

Troponin was isolated from the abdominal muscle of the American lobster (Homarus americanus) by essentially the same method as used for akazara scallop troponin [J. Biol. Chem. 261, 16749-16754 (1986)]. The thus isolated troponin together with lobster tropomyosin confers high Ca2(+)-sensitivity to rabbit reconstituted actomyosin. The troponin consists of components having Mr of about 42,000, 32,000, 30,000, and 17,000, but not the Mr 52,000-59,000 component previously reported to be present in several crustacean troponins. These troponin components were separated from each other by DEAE-Toyopearl column chromatography in the presence of 6 M urea. The Mr 17,000 component was further separated into one major and two minor components by the same chromatography, but each of them was confirmed to be a Ca2+ binding component, TnC. The Mr 32,000 and 30,000 components were both regarded as inhibitory subunits, TnIs, since the Mg-ATPase activity of actomyosin in the presence of tropomyosin was strongly inhibited by the addition of the components, and the inhibition was reversed by the further addition of TnC. Finally, the Mr 42,000 component was regarded as TnT, since this component formed stoichiometic complex with TnC and TnI, and was indispensable for Ca2+ regulation of the actomyosin-tropomyosin system.

Isolation of troponin-tropomyosin-containing thin filaments from ascidian smooth muscle

1. 1. The fine structure of mantle of ascidian was investigated with an electron microscope and was revealed to be similar in type to smooth muscle. 2. 2. Native thin filaments which contain actin, tropomyosin and troponin were isolated from the ascidian smooth muscle. 3. 3. The interaction of the ascidian thin filaments with rabbit myosin was strongly dependent on the presence of calcium ions. 4. 4. Troponin-tropomyosin complex and troponin, which provided rabbit reconstituted actomyosin with the responsiveness to calcium ions, were obtained from the ascidian thin filaments.

Cooperative interactions between the contractile proteins of cardiac and skeletal muscle

The calcium activation of the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cardiac actomyosin reconstituted from bovine cardiac myosin and a complex of actin-tropomyosin-troponin extracted from bovine cardiac muscle at 37°C was studied and compared with similar proteins from rabbit fast skeletal muscle. The proteins of the actin complex were identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Half-maximal activation of the cardiac actomyosin was seen at a calcium concentration of 1.2 ± 0.002 (S.E. of mean) μM. A hybridized reconstituted actomyosin made with cardiac myosin and the actin-tropomyosin-troponin complex extracted from rabbit skeletal muscle was also activated by calcium but the half-maximal value was shifted to 0.65 ± 0.02 (S.E. of mean) μM Ca 2+. Homologous rabbit skeletal actomyosin showed half-maximal activation at 0.90 ± 0.01 (S.E. of mean) μM Ca 2+ and the value for a hybridized actomyosin made with rabbit skeletal myosin and the actin-complex from cardiac muscle was found at 1.4 ± 0.03 (S.E. of mean) μM Ca 2+ concentration. Kinetic analysis of the Ca 2+-activated ATPase activity of reconstituted bovine cardiac actomyosin indicated some degree of cooperativity with respect to calcium. Double reciprocal plots of reconstituted actomyosins made with bovine cardiac actin complex were curvilinear and significantly different than those of reconstituted actomyosins made with the rabbit fast skeletal actin complex. The Ca 2+-dependent cooperativity was of a mixed type as determined from Hill plots for homologous reconstituted bovine cardiac and rabbit fast skeletal actomyosin. The results show that cooperative interactions in reconstituted actomyosins were greater when the actin-tropomyosin-troponin complex was derived from cardiac than skeletal muscle.

Myosin and actomyosin from human skeletal muscle.

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (+/-2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37 degrees C. Activation of the Mg-ATPase activity of Ca2+-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 muM Ca2+ concentration (CaEGTA binding constant equals 4.4 - 10(5) at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6-9 range, the Ca2+-ATPase activity of the subfragment 1 was 1.8- and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6-10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca2+-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca2+ is similar to that of rabbit fast or slow muscle.

Calcium sensitivity of actomyosin ATPase: its modification by substitution of myosin sulfhydryl groups.

SH group substitution by DTNB enabled natural actomyosin to split ATP (in the prescence of Mg2+) also in the absence of Ca2+, when assayed at low ionic strength. At higher KCl concentrations the ATPase activity of SH group substituted actomyosin was still Ca-dependent. Addition of unsubstituted myosin to natural actomyosin whose SH groups had been substituted increased the ATPase activity. This increase was Ca-insensitive indicating that SH group substitution of myosin in actomyosin can make the interaction of additional myosin molecules Ca-independent. In natural actomyosin Ca-insensitivity of ATPase activity was attained at a lower degree of SH group substitution when substitution was performed in the presence of EDTA. The part of ATPase activity which still remained Ca-sensitive after DTNB treatment could be activated by lower concentrations of free Ca2+ than the Ca-sensitive ATPase of untreated actomyosin. In reconstituted actomyosin the Ca-sensitivity of ATPase activity could more easily be reduced when the myosin-actin ratio was high. For demonstrating remaining Ca-sensitivity in SH group substituted reconstituted actomyosin more tropomyosin-troponin was needed than for sensitizing unsubstituted actomyosin to Ca2+. The similarities between the ATPase acitivity of SH group substituted actomyosin on the one hand and that of actomyosin at low concentrations of ATP on the other hand suggest that SH group substitution modifies actin-myosin interaction in a similar way as does nucleotide-free myosin (rigor myosin).

α-Actinin from red and white porcine muscle

Properties of purified α-actinin prepared from red and white sections of porcine semitendinosus muscle were compared. Although Z-lines were 2.25 times wider in red semitendinosus than in white semitendinosus, identical yields of α-actinin were obtained from these two sections of the semitendinosus. Since α-actinin is located in the Z-line, this result indicates that the Z-line contains proteins in addition to α-actinin. Reconstituted actomyosin prepared from red semitendinosus had a lower Mg 2+-modified ATPase activity and a slower rate of turbidity increase than reconstituted actomyosin made from white semitendinosus; this result and the fact that red semitendinosus had wider Z-lines than the white semitendinosus confirmed that red semitendinosus contained a predominance of red fibers, whereas the white semitendinosus contained a predominance of white fibers. Red α-actinin contained more aspartic acid than white α-actinin; this difference was mainly responsible for red α-actinin having 17 more negatively charged amino acids per 1000 total amino acids than white α-actinin. Because of this difference in negatively charged, amino acid side chains, red α-actinin could be separated from white α-actinin by DEAE-cellulose chromatography or polyacrylamide gel electrophoresis. Red α-actinin did not differ from white α-actinin in sedimentation pattern (both 6.1 S), in circular dichroic spectra (both about 59% α-helical), in rate of digestion by trypsin, or in ability to increase the Mg 2+-modified ATPase activity or rate of turbidity increase in suspensions of either red or white reconstituted actomyosin. The properties of porcine α-actinin are very similar to those of rabbit α-actinin, but the physiological role of α-actinin remains undetermined.

Purification and Properties of a Tropomyosin-containing Protein Fraction That Sensitizes Reconstituted Actomyosin to Calcium-binding Agents

Abstract A protein fraction that confers 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane (EGTA) sensitivity on reconstituted actomyosin made with highly purified actin and myosin has been prepared from actin extracted from the acetone-dried muscle powder at 25–37°. This protein fraction had effects upon reconstituted actomyosin that, in the absence of Ca++-binding agents, resembled those of tropomyosin made by classical methods in that the Mg++-activated adenosine triphosphatase activity was inhibited during the clearing phase and enhanced after superprecipitation. Unlike tropomyosin made by classical methods, this protein fraction potentiated an inhibitory action of EGTA upon the Mg++-activated ATPase activity of reconstituted actomyosin. In the presence of Mg++ and the protein fraction, EGTA appeared to prevent activation of myosin ATPase by actin. The EGTA-sensitizing protein fraction, which sedimented as a single symmetrical peak in the ultracentrifuge, had a sedimentation coefficient of 2.66 S, a value similar to that of tropomyosin. The dependence of specific viscosity upon ionic strength was similar for both. However, the intrinsic viscosity of the protein fraction was less than that of tropomyosin, and amino acid analyses revealed several significant differences between the two. The relatively high contents of proline and phenylalanine found in the EGTA-sensitizing protein fraction indicate that a previously undescribed protein was present in addition to tropomyosin. It is possible that this additional protein material was responsible for sensitization of actomyosin to Ca++-binding agents, but the possibility remains that this action was produced by a native form of tropomyosin.