Internal flexibility of cardiac myosins

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) and differential scanning calorimetry (DSC) were used to study the motional dynamics and segmental flexibility of cardiac myosins.Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with isothiocyanate- or maleimide-based probe molecules at the reactive sulfhydryl sites (Cys-697 and Cys-707) of the motor domain. The maleimide probe molecules attached to human cardiac myosin rotated with an effective rotational correlation time of 33 ns which was at least eight times shorter than the rotational correlation time of the same label on skeletal myosin (260 ns). In the presence of MgADP and MgADP plus orthovanadate, flexibility changes in the multisubunit structure of myosins were detected, but this did not lead to changes of the overall rotational property of the myosin heads. Significant difference in the internal flexibility was detected on myosin samples isolated from ischemic tissue, the rotational correlation time decreased to 25 ns.DSC measurements supported the view that addition of nucleotides produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that there is an intersite communication between the nucleotide binding domain and the 20 kDa subunit where the reactive thiol sites are located.This work was supported by grants from the National Research Foundation (OTKA T 017099) and Ministry of Social Welfare (ETT 737/1993). The Bruker ESP 300 E spectrometer and the SETARAM Micro DSC-II used in the experiments were purchased with funds provided by the National Research Foundation Grants CO-123 and CO-272. The computer (PC-386) was supported by POPEX Ltd., Pécs, Hungary. The authors thank to Prof. Dr. K. Hideg (Central Research Laboratory) for providing the iodoacetamide spin label.     

应用胰凝乳蛋白酶消化法比较冬季和夏季鲢鱼肉肌球蛋白的构造特性

以夏季和冬季鲢为研究对象,利用胰凝乳蛋白酶能水解羧基端含芳香族氨基酸残基肽键的特性,根据其特异性酶切部位,结合电泳手段来分析肌球蛋白的内部构造差异性。结果表明,与夏季样品相比,冬季鲢的肌原纤维蛋白经酶切生成的肌球蛋白头部S-1较长,在高温下分子量为165 ku的重酶解肌球蛋白HMM容易被再降解成小片段的135 ku HMM,呈现出冬季肌球蛋白的结构不稳定性。在不同温度下加热夏季和冬季肌球蛋白,其ATPase失活速度和酶解肌球蛋白生成S-1的产生量的减少速度呈现一致性,说明酶解生成的S-1只来源于有活性的肌球蛋白。同时,冬季肌球蛋白热变性温度较夏季肌球蛋白要低6 ℃,表明冬季肌球蛋白的不稳定性。     

Model for processive movement of myosin V and myosin VI

Myosin V and myosin VI are two classes of two-headed molecular motors of the myosin superfamily that move processively along helical actin filaments in opposite directions. Here we present a hand-over-hand model for their processive movements. In the model, the moving direction of a dimeric molecular motor is automatically determined by the relative orientation between its two heads at free state and its head‘s binding orientation on track filament.This determines that myosin V moves toward the barbed end and myosin VI moves toward the pointed end of actin.During the moving period in one step, one head remains bound to actin for myosin V whereas two heads are detached for myosin VI: the moving manner is determined by the length of neck domain. This naturally explains the similar dynamic behaviours but opposite moving directions of myosin VI and mutant myosin V (the neck of which is truncated to only one-sixth of the native length). Because of different moving manners, myosin VI and mutant myosin V exhibit significantly broader step-size distribution than native myosin V. However, all the three motors give the same mean step size of -36nm (the pseudo-repeat of actin helix). All these theoretical results are in agreement with previous experimental ones.     

Simultaneous Measurement of Individual ATPase and Mechanical Reactions by a Single Myosin Molecule at Work

Based on techniques for single molecule imaging and nanomanipulation by optical tweezers, we have developed a new technique that allows simultaneous measurement of individual ATPase and mechanical reactions from a single myosin molecule during force generation. We show how the ATPase reaction couples to the mechanical reaction directly at the single molecule level. The results show that the myosin head can produce force even after releasing the bound nucleotide, probably ADP, suggesting that the chemical energy driven by ATP hydrolysis can be hysteretically stored in the myosin molecule. This view does not support a widely accepted hypothesis in which the force generation is tightly coupled to ligand dissociation.This paper was originally presented as an invited paper at the seventh Meeting on Near Field Optics, which was held on July 1, 1998 at Nagoya University, Nagoya, organized by the Research Group on Near Field Optics of the Optical Society of Japan, an affiliate of the Japan Society of Applied Physics.     

分子马达与布朗马达

综述了有关分子马达,主要是肌球蛋白马达和动蛋白马达的实验研究进展情况,并对理论模型,特别是近年来广为流行的布朗马达模型作了介绍和评论。最后展望了这一领域的发展前景及其所面临的挑战性问题。     

Collective alignment of polar filaments by molecular motors

We study the alignment of polar biofilaments, such as microtubules and actin, subject to the action of multiple molecular motors attached simultaneously to more than one filament. Focusing on a paradigm model of only two filaments interacting with multiple motors, we were able to investigate in detail the alignment dynamics. While almost no alignment occurs in the case of a single motor, the filaments become rapidly aligned due to the collective action of the motors. Our analysis shows that the alignment time is governed by the number of bound motors and the magnitude of the motors’ stepping fluctuations. We predict that the time scale of alignment is in the order of seconds, much faster than that reported for passive crosslink-induced bundling. In vitro experiments on the alignment of microtubules by multiple-motor covered beads are in qualitative agreement. We also discuss another mode of fast alignment of filaments, namely the cooperation between motors and passive crosslinks.     

Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.     

Method for Enhanced Separation of Cardiac Myosin Heavy Chain Isoforms by Nongradient SDS-PAGE

Abstract

Current methods of SDS-PAGE for cardiac myosin heavy-chain (MHC) isoforms are complex and require more than 24 h. The aim of the current study was to improve the methodology of gel electrophoresis for faster and efficient separation of MHC isoforms. Rat ventricle and soleus tissues were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using tris-glycine buffer. Matrix-assisted laser desorption ionization (MALDI)–time of flight (TOF)–mass spectra (MS) of protein bands was done to identify α,β-MHC. Clear separation of α,β-MHC isoforms on SDS-PAGE was achieved and identity was confirmed by MALDI-TOF-MS. The smaller gel system drastically reduced the total run time to 2 h. The present method provides a simple, quick, and efficient protocol for cardiac α,β-MHC separation.