Analysis of nucleotide myosin complexes in skeletal muscle fibres by DSC and EPR

The internal dynamics and thermal unfolding of fibre bundles prepared from rabbit psoas muscle has been studied in the presence of nucleotides by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Using ADP, adenosine 5'-triphosphate (ATP), AMP.PNP and inorganic phosphate analogue orthovanadate (V(i)), AlF(4)(-) and BeF(3)(-), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibres. In the main transition of the DSC pattern, three overlapping endotherms were detected in rigor, four in strongly as well as weakly binding state of myosin to actin. Deconvolution procedure showed that the transition temperature of 67.5 degrees C was the same for rigor and strong binding state of myosin. In contrast, nucleotide binding induced shift of the melting temperatures of 52 degrees C and 67.5 degrees C, appeared a new fourth peak at 74 and 77 degrees C and produced changes in the calorimetric enthalpies. The changes of the parameters of the peak functions suggest global rearrangements of the internal structure in myosin heads in the intermediate states. In the presence of ADP or ATP plus phosphate analogue orthovanadate or beryllium fluoride, aluminium fluoride, the conventional EPR spectra of spin-labeled muscle fibres showed large changes in the ordering of the probe molecules, and a new distribution of spin labels appeared. ATP plus orthovanadate induced the orientation disorder of myosin heads; the random population of spin labels gave evidence of large local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus phosphate analogues corresponding to weakly binding state of myosin to actin.     

Theoretical studies on capillary microviscometry of skeletal muscle actin

For improving Ostwald's viscometry, which is time-consuming and requires a relatively large volume of specimen to determine viscosity, we developed a capillary microviscometric method with an appropriate mathematical model, and have compared this method with Ostwald's method.     

Tetrahymena actin: copolymerization with skeletal muscle actin and interactions with muscle actin-binding proteins

We have previously shown that actin from Tetrahymena pyriformis has a very divergent primary structure (Hirono, M., Endoh, H., Okada, N., Numata, O., & Watanabe, Y. (1987) J. Mol. Biol. 194, 181-192) and that though it shares essential properties with skeletal muscle actin, it does not interact at all with phalloidin or DNase I (Hirono, M., Kumagai, Y., Numata, O., & Watanabe, Y. (1989) Proc. Natl. Acad. Sci. U.S. 86, 75-79). In this study, we investigated the copolymerization of this actin with skeletal muscle actin by direct observation of the heteropolymers formed from the two actins by means of electron microscopy. We also examined the binding of actin-binding proteins from skeletal muscle or smooth muscle to Tetrahymena actin by means of a cosedimentation assay. The results show that (i) Tetrahymena actin copolymerizes with skeletal muscle actin and that (ii) muscle myosin subfragment 1 binds to it in the absence of ATP, like skeletal muscle actin. However, it was also shown that (iii) muscle alpha-actinin hardly binds to Tetrahymena actin and that (iv) muscle tropomyosin does not bind to it at all. The results show that Tetrahymena actin has both properties similar and dissimilar to those of skeletal muscle actin.     

The interaction of hemin with skeletal muscle actin

The ability of actin to interact with hemin was studied. It was found that the Soret absorption band of hemin changes in the presence of actin and that hemin is capable of quenching the fluorescence intensity of actin. These findings were indicative of hemin binding to actin. The binding constant for the high affinity site was calculated to be 5.3 X 10(6) M-1. The amounts of native G- and F-actin were estimated by their DNAase I inhibition activity. It was observed that the binding of hemin to G-actin is followed by a slow decrease in the ability of actin to inhibit DNAase I activity and to polymerize upon addition of salts. Binding of hemin to F-actin resulted in a gradual depolymerization of the filaments, to an inactivated form, as expressed by a reduction in the ability of hemin-bound F-actin to inhibit DNAase I activity in the absence as well as in the presence of guanidine-HCl. Electron microscopy studies further corroborated these findings by demonstrating that: (1) hemin-bound G-actin failed to show formation of polymers when salts were added; (2) a marked reduction in the amount of actin polymers was observed in the specimens examined 24 h after mixing with hemin. It is suggested that the elevated amounts of free hemin formed under pathological conditions, might be toxic to cells by interfering with actin polymerization cycles.     

BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development

The actin cytoskeleton is an important regulator of cell expansion and morphogenesis in plants. However, the molecular mechanisms linking the actin cytoskeleton to these processes remain largely unknown. Here, we report the functional analysis of rice (Oryza sativa) FH5/BENT UPPERMOST INTERNODE1 (BUI1), which encodes a formin-type actin nucleation factor and affects cell expansion and plant morphogenesis in rice. The bui1 mutant displayed pleiotropic phenotypes, including bent uppermost internode, dwarfism, wavy panicle rachis, and enhanced gravitropic response. Cytological observation indicated that the growth defects of bui1 were caused mainly by inhibition of cell expansion. Map-based cloning revealed that BUI1 encodes the class II formin FH5. FH5 contains a phosphatase tensin-like domain at its amino terminus and two highly conserved formin-homology domains, FH1 and FH2. In vitro biochemical analyses indicated that FH5 is capable of nucleating actin assembly from free or profilin-bound monomeric actin. FH5 also interacts with the barbed end of actin filaments and prevents the addition and loss of actin subunits from the same end. Interestingly, the FH2 domain of FH5 could bundle actin filaments directly and stabilize actin filaments in vitro. Consistent with these in vitro biochemical activities of FH5/BUI1, the amount of filamentous actin decreased, and the longitudinal actin cables almost disappeared in bui1 cells. The FH2 or FH1FH2 domains of FH5 could also bind to and bundle microtubules in vitro. Thus, our study identified a rice formin protein that regulates de novo actin nucleation and spatial organization of the actin filaments, which are important for proper cell expansion and rice morphogenesis.     

Interaction in vitro of scallop muscle arginine kinase with filamentous actin.

Scallop muscle arginine kinase binds to F-actin from mollusc and rabbit muscle in vitro. One site of interaction appears to be located in residues 305-325 of a C-terminal fragment (residues 285-375) of actin. The binding is hindered in the presence of arginine, Mg(2+)-ADP and NO3-, which form a dead-end complex with the enzyme. F-actin inhibits the enzyme activity non-competitively with respect to Mg(2+)-ATP. As a function of arginine concentration, the inhibition is of the mixed type, where Km is affected more than Vmax.     

Interaction of lactate dehydrogenase with skeletal muscle troponin

Rabbit muscle troponin complex covalently bound to CNBr-activated Sepharose 4B was shown to interact with soluble lactate dehydrogenase with a stoichiometry of 2 mol lactate dehydrogenase/mol of troponin. The presence of Ca2+ influenced the strength of association (the KD values of 0.73 and 2.3 microM were determined in the presence of 200 microM EGTA or 100 microM Ca2+, respectively). In the absence of Ca2+, the affinity of lactate dehydrogenase to troponin was strongly pH-dependent, reaching a maximum in the region of pH 6.0-7.0. No change of catalytic activity was observed as a result of interaction between lactate dehydrogenase and troponin, the enzyme appeared capable of functioning in the bound form.     

Interaction of ADP-ribosylated actin with actin binding proteins.

Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin beta(4), cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.     

Interaction between rabbit muscle actin and several chaetoglobosins or cytochalasins.

The mold metabolites chaetoglobosins Ch-A, B, C, E, F, and J exert, as do the cytochalasins CB, CD, CE, and CG, enhancing effects of various strength on the polymerization of rabbit muscle G-actin. The polymers formed differ widely in their viscosity, Ch-B and Ch-J leading to the least viscous actins. Equal states of viscosity are arrived at by interaction of F-actin with the respective drugs. There is no correlation between the ATP hydrolyzing activity of F-actin elicited by the various cytochalasins and their influence on the viscosity.     

The formin mDia2 stabilizes microtubules independently of its actin nucleation activity

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset of MTs oriented toward the direction of migration. Although mDia nucleates actin filaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mutants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization-impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution-induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, respectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nucleation activity.     

Ca2+ control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation

We elucidated the mechanism by which gelsolin, a Ca2+-dependent regulatory protein from lung macrophages, controls the network structure of actin filaments. In the presence of micromolar Ca2+, gelsolin bound Ca2+. The Ca2+-gelsolin complex reduced the apparent viscosity and flow birefringence of F-actin and the lengths of actin filaments viewed in the electron microscope. However, concentrations of gelsolin causing these alterations did not effect proportionate changes in the turbidity of actin filament solutions or in the quantity of nonsedimentable actin as determined by a radioassay. From these findings, we conclude that gelsolin shortens actin filaments without net depolymerization. Such an effect on the distribution of actin filament lengths led to the prediction that low concentrations of gelsolin would increase the critical concentration of actin-binding protein required for incipient gelation of actin filaments in the presence of Ca2+, providing an efficient mechanism for controlling actin network structure. We verified the prediction experimentally, and we estimated that the Ca2+-gelsolin complex effectively breaks the bond between actin monomers in filaments with a stoichiometry of 1:1. The effect of Ca2+-gelsolin complex on actin solation was rapid, independent of temperature between 0 degrees and 37 degrees C, and reversed by reducing the free Ca2+ concentration.     

Interaction of calcium and local anesthetics with skeletal muscle microsomes

The influence of Ca⁺⁺, several drugs, and pH on the binding of Ca⁺⁺ by skeletal muscle microsomes was studied in vitro. A mass-law graphic analysis revealed the presence of three distinct species of Ca⁺⁺-binding sites in the microsomes, and the binding at only one of these sites was antagonized by local anesthetics and quinidine. These drugs also decreased the maximum Ca⁺⁺-binding capacity of the microsomes. Caffeine and ouabain exerted no effect on the binding at any of the sites. Procaine was also bound by microsomes, and this binding was antagonized by Ca⁺⁺, which also decreased the maximum procaine-binding capacity of microsomes. The sites that bind procaine and Ca⁺⁺ are not identical because the maximum-binding capacities of the interacting sites are distinctly different. The influence of pH on the ability of drugs to antagonize Ca⁺⁺ binding indicates that the displacing activity increases as the percentage of the drug in the nonionized form increases. All of the data obtained in the above studies are consistent with the interpretation that quinidine and local anesthetics of the procaine type noncompetitively antagonize the binding of Ca⁺⁺ by microsomes. The pharmacological significance of a noncompetitive interaction may be related to the property of local anesthetics and quinidine to increase contractile tension in skeletal muscle rather than to their ability to stabilize the cell membrane.     

Interaction of thymosin beta 4 with muscle and platelet actin: implications for actin sequestration in resting platelets.

Quantitative measurements of the interactions of T beta 4 with muscle actin suggest that its only physiological role is monomer sequestration. T beta 4 forms a 1:1 complex with monomeric actin under physiological salt conditions. Its Kd for actin is not affected by calcium. T beta 4 binds only to actin monomers and not to filament ends or alongside the filament. T beta 4-actin complexes do not elongate actin filaments at either the barbed or the pointed end, and, unlike actobindin, T beta 4 does not specifically suppress the nucleation of polymerization. We assessed the fraction of monomeric actin that can be sequestered by T beta 4 in resting platelets. This was done on the basis of (a) its Kd of 0.4-0.7 microM for platelet actin, which had been prepared by a newly devised simpler method, and (b) the values for the concentrations of monomeric actin and of T beta 4 which we measured as 280 and 560 microM, respectively. Using the higher Kd value of 0.7 microM, the T beta 4-complexed actin is calculated to be between 70 and 240 microM, depending on the steady-state free G-actin concentration. This may vary from 0.1 to 0.5 microM, the critical concentrations for uncapped and for fully barbed-end-capped actin filaments. If the Kd in the platelet is the same as in vitro, most of the sequestered actin would be bound to T beta 4 if more than 95% of the actin filaments are capped at their barbed ends in resting platelets.     

Interaction of bacterial flagellum filaments with skeletal muscle myosin

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+ -ATP. Addition of BFF inhibited both K+ -EDTA- and Ca2+ -ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+ -ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.     

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA

We compare a recombinant cDNA plasmid (pAF81) complementary to a fetal skeletal muscle actin mRNA with a plasmid (pAM91) complementary to the actin mRNA expressed in adult skeletal muscle. The two mRNAs are significantly diverged in silent nucleotide positions; they are coexpressed in fetal skeletal muscle, and in differentiating muscle cell cultures their accumulation begins coordinately. The sequence of pAF81 shows that the amino acid sequence of mouse fetal skeletal muscle actin is almost identical to that of adult bovine cardiac actin. Hybridization of pAF81 to RNA from different mouse tissues shows that fetal skeletal muscle actin mRNA is very homologous or identical to fetal and adult cardiac actin mRNA. Only one gene homologous to pAF81 is detected on blots of restricted mouse DNA. We conclude that this gene must be expressed both in fetal skeletal muscle and in fetal heart. Whereas mRNA transcribed from this gene is the major actin mRNA species in adult heart, it is present in low amounts, if at all, in adult skeletal muscle.     

Extra actin filaments at the periphery of skeletal muscle myofibrils.

Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the myofibril. Decoration with myosin subfragment 1 shows that these are actin filaments.     

Interaction of actin with the capping protein, CapZ from sea bass (Dicentrarchus labrax) white skeletal muscle

We have compared the functional properties of CapZ from fish white skeletal muscle with those of CapZ from chicken muscle. CapZ is a heterodimer, which enhances actin nucleation and inhibits the depolymerization process by binding to the barbed ends of microfilaments. Here, we report the interaction of CapZ not only with F-actin, but also with monomeric actin. The affinity of sea bass CapZ for G-actin estimated by enzyme-linked immunosorbent assay (ELISA) was in the μM range. This association was PIP₂ dependent. Binding contacts with the barbed end of actin were delimited by both ELISA and fluorescence approaches. One site (actin sequence 338–348) was located in a helical region of the subdomain 1, region already implicated in the interaction with other actin binding proteins such as gelsolin. Another site implicates the C-terminal region (sequence 360–372) of actin. Finally, the partial competition of antibodies directed against CapZ α or β-subunits towards CapZ interaction with actin filaments suggests both subunits participate in the complex with actin.