The Chromatography of “Myosin B” Prepared from Minced Rabbit Muscle on Triethylaminoethylcellulose

The chromatography on Cellex D DEAE-SF (Bio-Rad Lab.) or TEAE-cellulose (Serva) equilibrated against 0.28 m KCl solution containing 0.02 m tris-HCl buffer at pH 8.0 was found to be suitable for the refinement of myosin B.

The ultraviolet absorption spectrum and ATPase activity of the eluted fractions showed that “myosin B” was fractionated and purified by this technique, especially by the preferential removal of the fraction suggested as ribonucleic acid related substance.

The chromatography may provide the effective way to investigate changes of “myosin B” during aging of meat.     

The Effect of Experimental Hyperthyroidism on Characteristics of Actin–Myosin Interaction in Fast and Slow Skeletal Muscles

The molecular mechanism of the failure of contractile function of skeletal muscles caused by oxidative damage to myosin in hyperthyroidism is not fully understood. Using an in vitro motility assay, we studied the effect of myosin damage caused by oxidative stress in experimental hyperthyroidism on the actin–myosin interaction and its regulation by calcium. We found that hyperthyroidism-induced oxidation of myosin is accompanied by a decrease in the sliding velocity of the regulated thin filaments in the in vitro motility assay, and this effect is increased with the duration of the pathological process.     

New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response

ConclusionsWe identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain.     

The Changes of “Myosin B” during Storage of Rabbit Muscle

The process of the denaturation of “myosin B” solution was studied by the measurement of ATPase activity, SH groups, sedimentation behaviour and flow birefringence. When “myosin B” solution was stored at lower temperature, lower pH or higher ionic strength, the interaction between myosin A and actin became less strong, and further storage brought about an irreversible dissociation.

The condition for measuring Mg-modified ATPase activity of “myosin B” at low ionic strength was explained in the relation with superprecipitation.     

The Changes of “Myosin B” during Storage of Rabbit Muscle

The irreversibility of the dissociation of “myosin B” stored in 0.6 m KCl at pH 5.7 and 3°C was attributed to the rapid denaturation of F-actin dissociated from “myosin B”

F-Actin was less stable than myosin A, in 0.18~0.60 m KCl at pH 5.7 and temperatures between 0 ~3°C.

The decrease in the ability of F-actin to bind with myosin A was slightly dependent on storage temperature, and there was no apparent relation with the decrease in the solubility.

A hypothetical scheme for F-actin denaturation was proposed.     

Investigations of Molecular Mechanisms of Actin–Myosin Interactions in Cardiac Muscle

The functional characteristics of cardiac muscle depend on the composition of protein isoforms in the cardiomyocyte contractile machinery. In the ventricular myocardium of mammals, several isoforms of contractile and regulatory proteins are expressed–two isoforms of myosin (V1 and V3) and three isoforms of tropomyosin chains (α, β, and κ). Expression of protein isoforms depends on the animal species, its age and hormonal status, and this can change with pathologies of the myocardium. Mutations in these proteins can lead to cardiomyopathies. The functional significance of the protein isoform composition has been studied mainly on intact hearts or on isolated preparations of myocardium, which could not provide a clear comprehension of the role of each particular isoform. Present-day experimental techniques such as an optical trap and in vitro motility assay make it possible to investigate the phenomena of interactions of contractile and regulatory proteins on the molecular level, thus avoiding effects associated with properties of a whole muscle or muscle tissue. These methods enable free combining of the isoforms to test the molecular mechanisms of their participation in the actin–myosin interaction. Using the optical trap and the in vitro motility assay, we have studied functional characteristics of the cardiac myosin isoforms, molecular mechanisms of the calcium-dependent regulation of actin–myosin interaction, and the role of myosin and tropomyosin isoforms in the cooperativity mechanisms in myocardium. The knowledge of molecular mechanisms underlying myocardial contractility and its regulation is necessary for comprehension of cardiac muscle functioning, its disorders in pathologies, and for development of approaches for their correction.     

The Changes of “Myosin B” during Storage of Rabbit Muscle

The results of sedimentation studies revealed that “myosin B” extracted from rigor muscle was different from myosin B from fresh muscle; the former contained less contaminating myosin A and less main component but more heavy component than the latter did. The proportion of constitutional myosin A to actin in the former was less than that in the latter.

“Myosin B” from post-rigor muscle was very similar to that from rigor muscle in the sedimentation behaviours.

The extrapolated sedimentation coefficient fresh muscle was 99S.     

Two Classes of Myosin Inhibitors,Para-nitroblebbistatin and Mavacamten,Stabilize β-Cardiac Myosin in Different Structural and Functional States

In addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional relaxed and the super-relaxed states are widely assumed to correspond to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin, respectively. Disruption of the myosin SRX population is an emerging model in different heart diseases, such as hypertrophic cardiomyopathy, which results in excessive muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examined the structure–function relationships of two myosin ATPase inhibitors, mavacamten and para-nitroblebbistatin, and found that binding of mavacamten at a site different than para-nitroblebbistatin populates myosin into the SRX state. Para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps much more inhibited, ‘ultra-relaxed’ state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations in human β-cardiac heavy meromyosin that diverge significantly from the hypothetical open and closed states, and furthermore, mavacamten treatment causes greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and para-nitroblebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing states.     

Basis for the Isoform-specific Interaction of Myosin Phosphatase Subunits Protein Phosphatase 1c �� and Myosin Phosphatase Targeting Subunit 1

Myosin II association with actin, which triggers contraction, is regulated by orchestrated waves of phosphorylation/dephosphorylation of the myosin regulatory light chain. Blocking myosin regulatory light chain phosphorylation with small molecule inhibitors alters the shape, adhesion, and migration of many types of smooth muscle and cancer cells. Dephosphorylation is mediated by myosin phosphatase (MP), a complex that consists of a catalytic subunit (protein phosphatase 1c, PP1c), a large subunit (myosin phosphatase targeting subunit, MYPT), and a small subunit of unknown function. MYPT functions by targeting PP1c onto its substrate, phosphorylated myosin II. Using RNA interference, we show here that stability of PP1c β and MYPT1 is interdependent; knocking down one of the subunits decreases the expression level of the other. Associated changes in cell shape also occur, characterized by flattening and spreading accompanied by increased cortical actin, and cell numbers decrease secondary to apoptosis. Of the three highly conserved isoforms of PP1c, we show that MYPT1 binding is restricted to PP1c β, and, using chimeric analysis and site-directed mutations, that the central region of PP1c β confers the isoform-specific binding. This finding was unexpected because the MP crystal structure has been solved and was reported to identify the variable, C-terminal domain of PP1c β as being the region key for isoform-specific interaction with MYPT1. These findings suggest a potential screening strategy for cardiovascular and cancer therapeutic agents based on destabilizing MP complex formation and function.     

Effects of Troponin T Cardiomyopathy Mutations on the Calcium Sensitivity of the Regulated Thin Filament and the Actomyosin Cross-Bridge Kinetics of Human β-Cardiac Myosin

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca²⁺-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca²⁺ sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca²⁺ induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin.     

Myosin II-dependent exclusion of CD45 from the site of Fcγ receptor activation during phagocytosis

Fcγ receptor (FcγR)-mediated phagocytosis requires myosin II activity. Here we show that myosin II contributes to FcγR activation and subsequent F-actin assembly at the nascent phagocytic cup. Inhibition of myosin II attenuates phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of FcγR and binding of Syk to the ITAM. Furthermore, FcγR clusters independently of myosin II activity at the phagocytic cup, from which the receptor-like protein tyrosine phosphatase CD45 is excluded depending on myosin II activity. These findings suggest that myosin II-dependent segregation of CD45 from FcγR facilitates phosphorylation of the ITAM and triggers phagocytosis.     

Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.     

Myosin V随机步长分布的动力学分析

Myosin V利用ATP水解所释放的自由能,朝肌动蛋白微丝正端作连续的定向运动,平均步长约为36nm。最近几年,诸多实验数据表明,myosin V步长并不固定为36nm,马达各步长值和相应步长出现概率的柱状图符合高斯分布;且在负载力大于2pN的情况下会出现“中间步长”和“后退步子”的现象。可根据已有实验数据,同时考虑马达在跃迁过程中所受的溶液摩擦阻力、常负载力和高斯随机力对其跃迁距离的影响,提出一种跃迁模型,并以此为基础对上述现象进行理论解释。     

Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ inter- acted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.     

The Role of GSK-3β Phosphorylation in the Regulation of Slow Myosin Expression in Soleus Muscle during Functional Unloading

Skeletal muscle myosin phenotype (i.e., the predominance in the muscle of a particular isoform or isoforms of myosin heavy chains (MyHC)) determines the properties of muscle, such as contraction speed and fatigue. The aim of this study was to identify the functional relationship between the decrease of the nitric oxide (NO) content, the GSK-3β phosphorylation (leading to the GSK-3β activation), the NFATc1 amount in the muscle nuclei, and the MyHC I(β) isoform expression in the rat soleus muscle under gravitational unloading. Male Wistar rats were divided into five groups: the vivarium control group; the group of animals with a 7-day hind limb suspension receiving placebo; the group of animals with a hind limb suspension receiving a NO donor (L-arginine); the group of animals with a hind limb suspension receiving a NO donor and a NO-synthase inhibitor (L-NAME); and the group of animals with a hind limb suspension receiving a GSK-3β inhibitor. We have shown that a 7-day unloading leads to a NO content decrease in the soleus muscle, and this effect is prevented by L-arginine administration. In addition, administration of L-arginine blocks the GSK-3β phosphorylation decrease, NFATc1 export from the muscle nuclei, and MyHC I(β) expression decrease caused by unloading. The L-arginine effect in each case can be blocked by the NO-synthase inhibitor. Administration of the GSK-3β inhibitor prevents the unloading-induced NFATc1 export from the muscle nuclei and a decrease of the MyHC I(β) expression. The prevention of the MyHC I(β) expression decrease and the NFATc1 export from the nucleus by the selective GSK-3β inhibition confirms the hypothesis on the NO influence on the MyHC I(β) expression and the NFATc1 export from the nucleus via the GSK-3β phosphorylation decrease. Thus, the NO level decrease in the rat soleus muscle in unloading leads to the GSK-3β activation, which in turn, promotes the NFATc1 export from the nucleus and stabilization of the fast myosin phenotype.     

β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar¹,Ile⁴,Ile⁸]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Myosin structure: does the tail wag the dog?

Previous crystal structures of the myosin head have shown two different conformations, postulated to be the beginning and the end of the actomyosin power stroke. A new crystal structure reveals a dramatically different conformation; but how does this conformation fit into the force-generating cycle of actomyosin interactions?     

α-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine

The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and α-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether α-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with α-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to α-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with α-AP-2. siRNA silencing of α-AP-2 in cells significantly reduced CFTR endocytosis, further supporting α-AP-2 as the direct binding partner for CFTR. α-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell''s Waltzer myosin VI^(sv/sv) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI^(sv/sv) enterocytes. The findings support α-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.