Skeletal muscle myosin subfragment-1 induces bundle formation by actin filaments

As is well known, the light scattering intensity of F-actin solutions increases immediately upon formation of the rigor complex with subfragment-1 (S-1). We have found that after the initial rise in scattering, there is a further gradual increase in scattering (we call it "super-opalescence"). Fluorescence and electron microscopic observations of acto-S-1 solutions showed that super-opalescence results from formation of actin filament bundles once S-1 binds to F-actin. The actin bundles possessed transverse stripes with a periodicity of about 350 A, which suggested that in the bundles actin filaments are arranged in parallel register. The rate of the initial process of bundle formation (i.e. side-by-side dimerization) could be approximately estimated by measuring the initial rate of super-opalescence (V0). V0 had a maximum (V0m) at a molar ratio of S-1 to actin of 1;6-1;7, regardless of the actin concentration, pH (6-8.5), Mg2+ concentration (up to 5 mM), or ionic strength (up to 0.3 M KC1). Lower pH, higher Mg2+ concentration, and higher ionic strength increased V0m; V0 was proportional to the square of the actin concentration, regardless of the solution conditions.     

Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis

Cell division after mitosis is mediated by ingression of an actomyosin-based contractile ring. The active, GTP-bound form of the small GTPase RhoA is a key regulator of contractile-ring formation. RhoA concentrates at the equatorial cell cortex at the site of the nascent cleavage furrow. During cytokinesis, RhoA is activated by its RhoGEF, ECT2. Once activated, RhoA promotes nucleation, elongation, and sliding of actin filaments through the coordinated activation of both formin proteins and myosin II motors (reviewed in [1, 2]). Anillin is a 124 kDa protein that is highly concentrated in the cleavage furrow in numerous animal cells in a pattern that resembles that of RhoA [3-7]. Although anillin contains conserved N-terminal actin and myosin binding domains and a PH domain at the C terminus, its mechanism of action during cytokinesis remains unclear. Here, we show that human anillin contains a conserved C-terminal domain that is essential for its function and localization. This domain shares homology with the RhoA binding protein Rhotekin and directly interacts with RhoA. Further, anillin is required to maintain active myosin in the equatorial plane during cytokinesis, suggesting it functions as a scaffold protein to link RhoA with the ring components actin and myosin. Although furrows can form and initiate ingression in the absence of anillin, furrows cannot form in anillin-depleted cells in which the central spindle is also disrupted, revealing that anillin can also act at an early stage of cytokinesis.     

Cleavage of DNA-binding loops of actin by subtilisin prevent formation of a strong type of myosin binding with actin

In order to elucidate the role of DNA-binding loop of actin (amino acid residues 38-52) in mechanisms of muscle contraction, polarizational fluorimetry and ghost muscle fibers, containing thin filaments reconstructed by intact and subtilisin-cleaved G-actin were used. The thin filaments were modified by fluorescent probes rhodamin-phalloidin and 1,5-IAEDANS. Changes in orientation and mobility of the probes were considered as an indication of changes in actin conformation. The stage AM of ATP hydrolysis cycle was simulated. For this purpose, thin filaments were decorated by myosin subfragment-1 (S1) in the absence of nucleotide. It has been shown that S1 binding to actin is accompanied by changes in orientation and mobility of the fluorescent probes. For intact filaments, the changes of these parameters indicate the formation of a strong binding between S1 and actin. Cleavage of DNA-binding loop by subtilisin markedly inhibits this effect. The cleavage of actin by subtilisin has also been shown to diminish the changes in fiber birefringence, which takes place at the formation of F-actin-S1 complex in the muscle fiber. The spatial organization of the actin DNA-binding loop is suggested to play an important role in determining the character of myosin interaction with actin in the ATP hydrolysis cycle.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

Biological activity and the 3-methylhistidine content of actin and myosin

1. The 3-methylhistidine content of myosin varies according to muscle type. It is highest in myosin from white skeletal muscle and lower values are obtained from myosin of red skeletal and smooth muscle. 2. The 3-methylhistidine content of actin was similar in all of the types of muscle from which it was isolated. 3. The 3-methylhistidine of rabbit actin is localized in a single tryptic peptide that was readily modified during fractionation procedures. 4. Photo-oxidation studies indicated that the 3-methylhistidine residues are not essential for adeonsine triphosphatase and actin-combining activities of myosin. 5. During photooxidation G-actin lost completely the ability to polymerize to the F form before all the 3-methylhistidine was destroyed.     

Non-polymerizing long-pitch actin dimers that interact with myosin

The production of a soluble actomyosin complex would be a significant step toward elucidating molecular interactions responsible for biological movement. We took a systematic approach to producing soluble long-pitch actin dimers that are polymerization-deficient yet retain biological protein-protein interactions, including myosin binding. Actin mutant proteins and chemical crosslinking were combined with different polymerization inhibition strategies, including ADP-ribosylation, or the use of a polymerization-deficient actin mutant protein. While all of the long-pitch actin dimers retained interactions reflective of F-actin activity, each displayed different interactions with myosin. Myosin did not interact productively with long-pitch actin dimers capped with DNase-I, and led to filament formation of unmodified long-pitch actin dimers or dimers possessing a polymerization-deficient actin subunit. However, ADP-ribosylated long-pitch actin dimers interacted with myosin, giving this dimer great potential for producing a soluble actomyosin complex, which could greatly improve our understanding of the molecular basis of movement in cells, tissues, and organisms.     

Relation between cell activity and the distribution of cytoplasmic actin and myosin

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.     

Arp2 links autophagic machinery with the actin cytoskeleton

Macroautophagy involves lysosomal/vacuolar elimination of long-lived proteins and entire organelles from the cytosol. The process begins with formation of a double-membrane vesicle that sequesters bulk cytoplasm, or a specific cargo destined for lysosomal/vacuolar delivery. The completed vesicle fuses with the lysosome/vacuole limiting membrane, releasing its content into the organelle lumen for subsequent degradation and recycling of the resulting macromolecules. A majority of the autophagy-related (Atg) proteins are required at the step of vesicle formation. The integral membrane protein Atg9 cycles between certain intracellular compartments and the vesicle nucleation site, presumably to supply membranes necessary for macroautophagic vesicle formation. In this study we have tracked the movement of Atg9 over time in living cells by using real-time fluorescence microscopy. Our results reveal that an actin-related protein, Arp2, briefly colocalizes with Atg9 and directly regulates the dynamics of Atg9 movement. We propose that proteins of the Arp2/3 complex regulate Atg9 transport for specific types of autophagy.     

Isolation of concanavalin a caps during various stages of formation and their association with actin and myosin

Regions of plasma membrane of dictyostelium discoideum amoebae that contain concanavalin A (Con A)-receptor complexes are more resistant to disruption by Triton X-100. This resistance makes possible the isolation of Con A-associated membrane fragments in sufficient quantity and homogeneity to permit the direct biochemical and ultrastructural study of receptor-cytoskeletal interactions across the cell membrane. After specific binding of Con A to the cell surface, a large amount of the cell’s actin and myosin copurifies with the plasma membrane fragments. Myosin is more loosely bound to the isolated membranes that actin and is efficiently removed by treating membranes with ATP and low ionic strength. If cells are not lysed immediately after lectin binding, all of the Con A that is bound to the cell surface is swept into a cap in a process requiring metabolic energy. When cells are lysed at different stages of cap formation, the amount of actin and myosin that copurifies with the isolated membranes remains the same. Thick and thin filaments that are attached to the protoplasmic surface of the isolated membranes underlie lectin-receptor complexes during all stages of cap formation. Once the cap is complete, the amount of actin and myosin that tightly bound to the plasma membrane is concentrated into the cap along with the Con A-receptor complexes. These results suggest that the ATP-dependent sliding of membrane-associated actin and myosin filaments is responsible for the accumulation of Con A-receptor complexes into a cap on the cell surface.     

Immobilization of actin and myosin.

Using glutaric dialdehyde, the muscle proteins myosin, actin, actomyosin and heavy meromyosin subfragment-1 (S-1) have been immobilized on capron fibers. The ATPase activity of myosin and its capability to interact with actin have been preserved whereas the ATPase activity of its subfragment decreased significnatly. Immobilization on capron fibers changes the pH dependence of the ATPase activity of myosin and of S-1 shifting the maximum towards the acid zone (pH 5.5) and increases the thermal stability of the enzyme. Calcium ions produce a stimulatory effect on ATPase; Mg2+ions yield no effect on myosin and S-1 but enhance the activity in the case of immobilized actomyosin though to a lesser degree than the ions of Ca2+. Immobilized actin retains its ability to form actomyosin complex.     

Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity

Actin polymerization essential for endocytic internalization in budding yeast is controlled by four nucleation promoting factors (NPFs) that each exhibits a unique dynamic behavior at endocytic sites. How each NPF functions and is regulated to restrict actin assembly to late stages of endocytic internalization is not known. Quantitative analysis of NPF biochemical activities, and genetic analysis of recruitment and regulatory mechanisms, defined a linear pathway in which protein composition changes at endocytic sites control actin assembly and function. We show that yeast WASP initiates actin assembly at endocytic sites and that this assembly and the recruitment of a yeast WIP-like protein by WASP recruit a type I myosin with both NPF and motor activities. Importantly, type I myosin motor and NPF activities are separable, and both contribute to endocytic coat inward movement, which likely represents membrane invagination. These results reveal a mechanism in which actin nucleation and myosin motor activity cooperate to promote endocytic internalization.     

Factors influencing interaction of phosphorylated and dephosphorylated myosin with actin

The influence of various factors on the interaction of phosphorylated and dephosphorylated myosin with actin was examined. It was found that the difference between the values of specific activity of the two myosin forms of actin-stimulated Mg2+-ATPase is affected by changes in KCl, MgATP and actin concentration. The effect of increased pH on the differences in the rate of ATP hydrolysis by actomyosin containing phosphorylated myosin as compared with that of the dephosphorylated one, observed in the presence of EGTA, is abolished by addition of Ca2+. Tropomyosin strongly inhibits the actin-stimulated Mg2+-ATPase of phosphorylated myosin (by about 60%). The tropomyosin-troponin complex and native tropomyosin lowered the rate of ATP hydrolysis by actomyosin containing both phosphorylated and dephosphorylated myosin by about of 60% of the value obtained in the absence of those proteins. These results indicate that the change of negative charge on the myosin head due to phosphorylation and dephosphorylation of myosin light chains modulates the actin-myosin interaction at different steps of the ATP hydrolysis cycle. Phosphorylation of myosin seems to be a factor decreasing the rate of ATP hydrolysis by actomyosin under physiological conditions.     

Interaction of actin with myosin A and heavy meromyosin.

Ca2+ "free" actomyosin suspensions as well as actin heavy meromyosin (HMM) solutions in the presence of Ca2+ showed no contractile response (superprecipitation) and had low steady-state Mg2+-ATPase activity. Under the same experimental conditions both the enzymatic activity increased and contractile response was restored if the solubility of the proteins was depressed by the addition of polyethylene glycol 4000 (PEG-4000). The stability of the enzymatically active actomyosin or actin HMM complexes was 10 times lower in cleared solutions than in the insoluble actomyosin or actin HMM suspensions. It was concluded that soluble actomyosin or actin HMM solutions are inadequate test tube models for studying muscular contraction.     

Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.     

Interaction of myosin subfragment 1 with forms of monomeric actin

The ability of myosin subfragment 1 to interact with monomeric actin complexed to sequestering proteins was tested by a number of different techniques such as affinity absorption, chemical cross-linking, fluorescence titration, and competition procedures. For affinity absorption, actin was attached to agarose immobilized DNase I. Both chymotryptic subfragment 1 isoforms (S1A1 and S1A2) were retained by this affinity matrix. Fluorescence titration employing pyrenyl-actin in complex with deoxyribonuclease I (DNase I) or thymosin beta4 demonstrated S1 binding to these actin complexes. A K(D) of 5 x 10(-8) M for S1A1 binding to the actin-DNase I complex was determined. Fluorescence titration did not indicate binding of S1 to actin in complex with gelsolin segment 1 (G1) or vitamin D-binding protein (DBP). However, fluorescence competition experiments and analysis of tryptic cleavage patterns of S1 indicated its interaction with actin in complex with DBP or G1. Formation of the ternary DNase I-acto-S1 complex was directly demonstrated by sucrose density sedimentation. S1 binding to G-actin was found to be sensitive to ATP and an increase in ionic strength. Actin fixed in its monomeric state by DNase I was unable to significantly stimulate the Mg2+-dependent S1-ATPase activity. Both wild-type and a mutant of Dictyostelium discoideum myosin II subfragment 1 containing 12 additional lysine residues within an insertion of 20 residues into loop 2 (K12/20-Q532E) were found to also interact with actin-DNase I complex. Binding of the K12/20-Q532E mutant to the actin-DNase I complex occurred with higher affinity than wild-type S1 and was less sensitive to mono- and divalent cations.