Reconstitution of contractile actomyosin rings in vesicles.

One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.

Talin dissociates from RIAM and associates to vinculin sequentially in response to the actomyosin force.

Cells reinforce adhesion strength and cytoskeleton anchoring in response to the actomyosin force. The mechanical stretching of talin, which exposes cryptic vinculin-binding sites, triggers this process. The binding of RIAM to talin could regulate this mechanism. However, the mechanosensitivity of the talin-RIAM complex has never been tested. It is also not known whether RIAM controls the mechanosensitivity of the talin-vinculin complex. To address these issues, we designed an in vitro microscopy assay with purified proteins in which the actomyosin force controls RIAM and vinculin-binding to talin. We demonstrate that actomyosin triggers RIAM dissociation from several talin domains. Actomyosin also provokes the sequential exchange of RIAM for vinculin on talin. The effect of RIAM on this force-dependent binding of vinculin to talin varies from one talin domain to another. This mechanism could allow talin to biochemically code a wide range of forces by selecting different combinations of partners.

Effects of l-arginine on the physicochemical and gel properties of chicken actomyosin.

The objective of this study is to investigate the effects of l-arginine (Arg) on the physicochemical and gel properties of chicken actomyosin. The results showed that Arg increased the content of surface hydrophobicity and reactive sulfhydryl group of chicken actomyosin, but decreased storage modulus (G0). Also, Arg enhanced the first thermal transition temperature (TM1) but decreased the second thermal transition temperature (TM2). The addition of Arg favored to form a dense and uniform gel with the increased water holding capacity (WHC), strength and transverse relaxation time (T2). These results suggested that Arg may result in the formation of a uniform and continuous gel by changing the structural and thermal behavior of actomyosin in turn, ultimately contributing to the elevated WHC and strength. The results may provide new insight into the effects of Arg on the WHC and texture of meat products in the previous literatures.

Isolation of Cytokinetic Actomyosin Rings from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Cytokinesis is the final stage of cell division, through which cellular constituents of mother cells are partitioned into two daughter cells resulting in the increase in cell number. In animal and fungal cells cytokinesis is mediated by an actomyosin contractile ring, which is attached to the overlying cell membrane. Contraction of this ring after chromosome segregation physically severs the mother cell into two daughters. Here we describe methods for the isolation and partial purification of the actomyosin ring from the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which can serve as in vitro systems to facilitate biochemical and ultrastructural analysis of cytokinesis in these genetically tractable model systems.

Impact of zinc salts on heat-induced aggregation of natural actomyosin from yellow stripe trevally.

Impact of zinc sulphate (ZnSO(4)) and zinc chloride (ZnCl(2)) on heat-induced aggregation of natural actomyosin (NAM) extracted from yellow stripe trevally (Selaroides leptolepis) was investigated. In the presence of ZnSO(4) or ZnCl(2), the transition temperature (T(max)) of myosin shifted from 47.83 ± 0.30°C to 46.05 ± 0.36 and 46.49 ± 0.49°C, with the coincidental decreases in ΔH from 1.07 ± 0.03 J/g to 0.63 ± 0.02 and 0.67 ± 0.04 J/g, respectively (P<0.05). Additionally, Ca(2+)-ATPase activity of NAM decreased with increasing the concentrations of ZnSO(4) or ZnCl(2) during heating up to 40°C. During heating from 20 to 75°C, higher turbidity, surface hydrophobicity and disulphide bond formation were obtained in NAM added with ZnSO(4) or ZnCl(2) at temperatures ranging from 40 to 75°C, compared with the control. Nevertheless, a higher aggregation was found in NAM added with ZnSO(4,) compared with ZnCl(2.) Zeta potential (ζ) analysis suggested that the surface of NAM added with ZnSO(4) became less negatively charged, compared with that of ZnCl(2) counterpart. Transmission electron microscopy showed that the structure of NAM was highly interconnected, finer and denser when zinc salts, especially ZnSO(4) were incorporated. Therefore, ZnSO(4) could be used to induce aggregation of fish muscle proteins, thereby improving gelling property of fish mince or surimi.

[Effect of electromagnetic field of extremely low frequency on ATPase activity of actomyosin].

The Mg2+/Ca2+ and K(+)-ATPase actomyosin activity of rabbit skeletal muscle was evaluated by the Fiske-Subbarow method during a five-hour exposition of protein solutions in electromagnetic field of extremely low frequency of 8 Hz and 25 microT induction. The results of the study of the ATPase activity of actomyosin upon electromagnetic exposure have shown statistically significant changes that are characterized by a rather complex time dynamics. After 1, 2 and 4 hours of exposure of protein solutions the effect of ELF EMF exposure inhibits the ATPase activity compared to control samples, which are not exposed to the magnetic field. By the third and fifth hours of exposure to the electromagnetic field, there is a significant increase in the ATPase activity of actomyosin. It should be noted that a similar pattern of change in enzyme activity was universal, both for the environment by Mg2+ and Ca2+, and in the absence of these ions in the buffer. This can evidence for Ca(2+)-independent ways of the infuence of electromagnetic field (EMP) on biologic objects. In our opinion, the above effects are explained by EMP influence on the dynamic properties of actomyosin solutions, which are based on the processes of spontaneous dynamic formation of structure.

[Influence of variuos ultrasound regimens on oxide-modified actomyosin superprecipitation reaction from skeletal muscle of rabbit].

A comparative study of rabbit skeletal muscles oxide-modified actomyosin superprecipitation reaction in dependence on continuous and impulsive (2 ms) ultrasound regimens was studied. From the analyses of kinetic curves the effect of the value of superprecipitation (A(m) - A(0)), time t1/2, required for achievement of half of its value was determined, and the normalized maximal rate of this reaction V was also calculated. It is shown that the use of continuous ultrasound to oxide-modified actomyosin was associated with a significant decrease of superprecipitation relative to controls. However, pulsed ultrasound caused a significant increase in superprecipitation value except for the values (A(m) - A(0)) in the application of the intensity of 0.2 W/cm2. The oxide-modified actomyosin superprecipitation value under the effect of continuous and impulsive ultrasound at intensity 1 W/cm2 in relative to control and all other applied intensities decrease to the most extent. It is caused perhaps by thermal influence of ultrasound. In general, the data obtained give reason to assume that the effects of continuous and pulsed ultrasound on the reaction of oxide-modified proteins complex superpretsipitatsii identical.

[Effect of continuous and impulsive ultrasound on actomyosin superprecipitation reaction from rabbit skeletal muscles].

A comparative study of the effect of continuous and impulsive (2 ms) ultrasound regimes on actomyosin superprecipitation reaction of the rabbit skeletal muscles was carried out. From the obtained kinetic curves the value of superprecipitation (D(m) - D(00)), time t1/2, which required to achieve a half its value was determined, and the normalized maximal rate of this reaction V(n) was calculated as well. It is shown that continuous ultrasound with intensities 0.7 W/cm2 and impulsive ultrasound--of 0.4 W/cm2 produced the most pronounced effect (value of superprecipitation and V(n) were maximal). The actomyosin superprecipitation value under continuous and impulsive ultrasound with intensity 1 W/cm2 relative to control and all other applied intensities was most decreased. It is caused perhaps thermal influence of the ultrasound. Thus the obtained data give every reason to assume, that impulsive ultrasound causes changes of adaptive character in actomyosin because the interrupted regime of the ultrasound signal.

Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus.

Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested-actin, myosin heavy and light chain, and IQGAP-as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis.

Comparative studies on the biochemical characteristics of natural actomyosin isolated from PSE and normal pork.

Biochemical changes of natural actomyosin from fresh pale, soft, exudative (PSE) and normal pork were studied, and the effects of different storage temperatures and different incubation temperature and times on sample superprecipitation, total sulfhydryl (-SH) content, and ATP (adenosine triphosphate) sensitivity were investigated. The results demonstrated that ATPase activity and thermal stability of PSE actomyosin were lower than those of normal pork; and that PSE actomyosin had higher -SH content than that of normal pork at all incubation temperatures and times tested.

Evidence of a new type of protein-protein interaction: desensitized actomyosin blocks Ca(2+)-sensitivity of the natural one. A possible model for an intracellular signalling system related to actin filaments.

Actin filaments are certainly believed to function as an intracellular signalling system; however, this is not confirmed by direct evidence. We used a two-layer actomyosin gel with a concentration gradient of the troponin-tropomyosin complex (TT-complex, Ca(2+)-sensitive system) between the two layers. To prepare one layer of the system, natural actomyosin (nAM) rich in TT-complex was used. To prepare the second layer, we used desensitized actomyosin (dAM) without the complex. All experimental studies were made in medium with a low ionic strength. Two phenomena were observed: (1) dAM blocks Ca(2+)-sensitivity of nAM when the dAM weight portion in the system (as well as in mixed nAM + dAM suspension) reaches 40% and more; further increase of the dAM portion does not affect the Ca(2+)-sensitivity; (2) it was electrophoretically shown that a rapid diffusion of the TT-complex from nAM gel into the dAM gel took place. The apparent diffusion coefficient for the TT-complex in dAM gel is about (1-4).10(-4) cm2/sec, i.e. three orders higher than the same values for protein diffusion in water.

Nonspecific weak actomyosin interactions: relocation of charged residues in subdomain 1 of actin does not alter actomyosin function.

Yeast actin mutants with relocated charged residues within subdomain 1 were constructed so we could investigate the functional importance of individual clusters of acidic residues in mediating actomyosin weak-binding states in the cross-bridge cycle. Past studies have established a functional role for three distinct pairs of charged residues within this region of yeast actin (D2/E4, D24/D25, and E99/E100); the loss of any one of these pairs resulted in the same impairment in weak actomyosin interaction and in its function. However, the specificity of myosin interaction with these sites has not yet been addressed. To investigate this, we made and analyzed two new actin mutants, 4Ac/D24A/D25A and 4Ac/E99A/E100A. In these mutants, the acidic residues of the D24/D25 or E99/E100 sites were replaced with uncharged residues (alanines) and a pair of acidic residues was inserted at the N-terminus, maintaining the overall charge density of subdomain 1. Using the in vitro motility assays, we found that the sliding and force generation properties of these mutant actins were identical to those of wild-type actin. Similarly, actin-activated ATPase activities of the mutant and wild-type actins were also indistinguishable. Additionally, the binding of S1 to these mutant actins in the presence of ATP was similar to that of wild-type actin. These results show that relocation of charged residues in subdomain 1 of actin does not affect the weak actomyosin interactions and actomyosin function.

Alpha1-syntrophin has distinct binding sites for actin and calmodulin.

Overlay and co-sedimentation assays using recombinant alpha1-syntrophin proteins revealed that two regions of alpha1-syntrophin, i.e. aa 274-315 and 449-505, contain high-affinity binding sites for F-actin (Kd 0.16-0.45 microM), although only a single high-affinity site (Kd 0.35 microM) was detected in the recombinant full-length syntrophin. We also found that actomyosin fractions prepared from both cardiac and skeletal muscle contain proteins recognized by anti-syntrophin antibody. These data suggest a novel role for syntrophin as an actin binding protein, which may be important for the function of the dystrophin-glycoprotein complex or for other cell functions. We also found that alpha1-syntrophin binds calmodulin at two distinct sites with high (Kd 15 nM) and low (Kd 0.3 microM) affinity.

Expression and regulation of mutant forms of cardiac TnI in a reconstituted actomyosin system: role of kinase dependent phosphorylation.

When phosphorylated, the inhibitory subunit of troponin (TnI) causes a loss in calcium sensitivity and a decrease in actomyosin ATPase. To examine this process, we bacterially expressed wild type TnI and TnI mutants in which serine 22 and 23, a putative protein kinase A (PKA) site, and threonine 143, a putative protein kinase C (PKC) site, were replaced by alanine S22A/23A and TI43A. PKA dependent phosphorylation was approximately 90% reduced in the S22A/23A mutant and unaffected in T143A. PKC dependent phosphorylation was markedly reduced in T 143A relative both to a wild type construct and to S22A/23A, although some residual phosphorylation (likely at sites other than T143) was seen. The calcium sensitivity (i.e. inhibition of actomyosin ATPase in the presence of EGTA) and regulation of the reconstituted actomyosin system was preserved in the absence of phosphorylation using wild type TnI or either mutant. Calcium sensitivity was decreased by both PKA and PKC with the wild type TnI but was unaffected by PKA when the S22A/23A mutant was employed and by PKC when the T143A mutant was reconstituted. The calcium dependency of the ATPase curve was substantially right shifted when PKC phosphorylated wild type TnI was employed for regulation, and this was markedly attenuated when T143 A was reassociated (although a slight rightward shift and a reduction in maximal ATPase activity was still seen). These data confirm that phosphorylation of TnI by regulatory kinases plays a major role in the regulation of myofibrillar ATPase. The N-terminal serines (22 and 23) appear to be uniquely important for the PKA response whereas threonine 143 is involved in the PKC response although other residues may also have functional significance.

[Effect of temperature on superprecipitation of actomyosin of myometrial smooth muscle and reactions of ATP hydrolysis catalyzed by this protein]. / Vliianie temperatury na superpretsipitatsiiu aktomiozina gladkoi myshtsy miometriia i reaktsiiu gidroliza ATP, kataliziruemuiu étim belkom.

The experiments carried out on the preparation of myometrium actomyosin were carried out. The reaction of superprecipitation of this protein and ATP hydrolysis were studied as affected by temperature. For the both processes the value of temperature optimum is 45 degrees C. The value of activation energy Ea is equal to 20 +/- 3 and 23 +/- 3 kJ/mol for the reaction of superprecipitation and ATP hydrolysis, respectively. Preincubation of actomyosin (45-60 degrees C, 1-5 min) in the medium not including ions of Ca, Mg and ATP with following introduction of the components to this medium and ten-minute incubation of protein in it causes the inhibition of the both reactions at the incubation temperature 45 degrees C and their stimulation at the incubation temperature 55 and 60 degrees C. Data obtained are discussed with regard for the structure organization of actomyosin of the smooth muscle.

The effect of cross-linking of the two heads of porcine aorta smooth muscle myosin on its conformation and enzymic activity.

The two heads of porcine aorta smooth muscle myosin can be cross-linked by a disulfide bridge between the two 17-kDa essential light chains with 5,5'-dithiobis(2-nitrobenzoic acid) [Katoh, T., Tanahashi, K., Hasegawa, Y. & Morita, F. (1995) Eur. J. Biochem. 227, 459-465]. When the cross-linked myosin sample was visualized by rotary shadowing, the two heads of myosin molecules appeared predominantly to adhere to each other. The cross-linking of dephosphorylated myosin in the presence of ATP was greatly inhibited by a decrease in the concentration of NaCl from 0.4 M to 0.15 M, suggesting that the cross-linking of the two heads was suppressed in 10S myosin. However, the fraction of dephosphorylated myosin in a filamentous state at 0.1 M NaCl in the presence of 1 mM ATP was increased from 33% to 83% by the cross-linking. The cross-linking of the two heads might inhibit the formation of the 10S conformation, leading to the increase in the fraction of filamentous myosin. The filaments of the cross-linked myosin sample were visualized by electron microscopy and appeared morphologically similar to those of uncross-linked myosin. The ATPase activity of the cross-linked dephosphorylated myosin sample was more than three times as high as that of an uncross-linked control. The increase in the activity may be related to the increase in the fraction of filamentous myosin caused by the cross-linking. The ATPase activity of dephosphorylated myosin in the presence of actin was increased more than twofold by the cross-linking, but the activity of phosphorylated myosin was affected only slightly. The degree of phosphorylation-dependent regulation of actin-activated ATPase activity decreased with an increase in the degree of cross-linking and was extrapolated to zero at 100% cross-linking. Superprecipitation of acto-cross-linked dephosphorylated myosin was activated, while that of acto-cross-linked phosphorylated myosin was inhibited only slightly. These results suggest that the freedom of each head in myosin molecules may be required to keep the ATPase activity and superprecipitation of acto-dephosphorylated myosin low but not for keeping these activity levels high in acto-phosphorylated myosin.

Determination of the protein components of native thin filaments isolated from natural actomyosin: nebulin and alpha-actinin are associated with actin filaments.

A new method was developed for extracting natural actomyosin with full length connectin (titin) and nebulin from rabbit skeletal muscle. To determine the protein components of native thin filaments, thin filaments were isolated from natural actomyosin by sedimentation in a H2O/D2O/sucrose density gradient. Both alpha-actinin and nebulin were cosedimented with actin filaments, while connectin was not. This shows that the native thin filaments contained alpha-actinin and nebulin. This indicates that the native thin filaments were more complicated than synthetic filaments reconstituted from actin, tropomyosin, and troponin. It is known that synthetic filaments are less Ca2+ sensitive than native thin filaments. This difference in Ca2+ sensitivity may be due to the differences in components and/or the different structures of native and synthetic filaments. The newly developed methods described here for extracting natural actomyosin and for isolating native thin filaments are useful for addressing these important problems related to the structure and function of native thin filaments.

Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins.

Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described.

Purification and characterization of an 'actomyosin' complex from Escherichia coli W3110.

An 'actomyosin' complex was purified from Escherichia coli W3110 using selective precipitation. The complex contains three major components of 19.5, 18.5 and 17 kDa. The 19.5- and 17-kDa proteins were purified by electroelution, peptide mapped and N-terminally sequenced. The structural gene for the 17-kDa protein was found to have been previously identified in an operon containing several other genes including the essential lpxA, lpxB and dnaE. The possible function of the 17-kDa protein and the other 'actomyosin' components is discussed.

Regulation of actomyosin interactions in Limulus muscle proteins.

Contraction of striated muscle from Limulus polyphemus, the horseshoe crab, is regulated by both calcium binding to a troponin-tropomyosin-dependent thin filament array and a myosin light chain kinase-dependent phosphorylation of myosin. We have isolated myosin from Limulus striated muscle and examined how these two regulatory systems affect the sliding velocity of actin filaments over myosin, using an in vitro motility assay. Our results show that in the presence of ATP, Limulus myosin must be phosphorylated in order to move actin filaments. No movement was observed for actin filaments interacting with dephosphorylated Limulus myosin. Calcium was not required for actin movement. In contrast, when both troponin and tropomyosin are bound to actin filaments, calcium is required for the movement of actin filaments over phosphorylated myosin. These results demonstrate that the "off" state of either the thin filament or thick filament regulatory system is dominant and that for the movement to occur, both phosphorylated Limulus myosin and an activated troponin-tropomyosin system are required. Tropomyosin by itself increases the sliding velocity of actin filaments over phosphorylated Limulus myosin about 10-fold in a calcium-independent manner. Tropomyosins from turkey gizzard smooth muscle, bovine cardiac muscle, and Limulus muscle all have a profound effect in increasing the velocity. Troponin alone does not change the velocity. Partial sequences of the tryptic phosphopeptides of Limulus myosin regulatory light chains generated following the phosphorylation by gizzard myosin light chain kinase yield ATS(PO4)NVFAMFEQNQIA for 21 kDa and SGS(PO4)NVFSMFT for 31-kDa light chain.