微丝

生命在于运动,各类生物有机体都在进行运动。动物和人有肌肉,肌肉收缩引起运动。构成肌肉的是肌纤维,肌纤维内有肌原纤维,肌原纤维内有粗细两种纤维,粗纤维含有肌球蛋白,细纤维含有肌动蛋白,两种蛋白相互滑动,使肌肉进行收缩运动。肌球蛋白和肌动蛋白不但分布在肌肉细胞里,还广泛地分布在非肌肉细胞里。在非肌肉细胞里它们以微丝的形式存在。肌动蛋白在2M Mg~(++)或0.1M K~+存在下,在体布能形成微丝,或者以未聚合的肌动蛋白单体的形式存在。各类真核细胞都发现有微丝,动植物细胞的生长和细胞的有丝分裂,细胞质的流动,以及变形虫和粘菌的变形运动,这些都有微丝的功能。     

肌动蛋白,肌动蛋白结合蛋白质和细胞运动的研究进展

运动是生命细胞的基本特征之一。肌动蛋白、肌球蛋白和调节蛋白质参与细胞运动和肌肉收缩,籍以完成不同的生理功能。蛋白质的结构生物学、分子遗传学和体外运动分析的应用,阐明了肌动蛋白、肌球蛋白和一些结合蛋白质的氨基酸序列、结合功能域和原子结构,使运动和能量转换可在分子水平上进行探索,促进了运动蛋白质体系的运动及其调节机制和能量转换机制研究的发展。     

果蝇非常规肌球蛋白的结构与功能

肌球蛋白是一类重要的分子马达,可以将ATP水解产生的能量转化成动能,沿由肌动蛋白组成的细丝运动。肌球蛋白构成一个大的基因家族,在许多细胞活动中起着重要作用,包括肌肉收缩、胞内转运、听觉、视觉等。果蝇基因组有13种肌球蛋白基因,包括2种常规肌球蛋白和11种非常规肌球蛋白。本文综述了近年来果蝇非常规肌球蛋白的研究进展。     

肌球蛋白工作循环的一个新模型

分析总结关于分子马达肌球蛋白的最新研究结果,给出一个新的肌球蛋白工作循环的机械化学偶联模型.从新模型出发,用一组化学动力学方程描述肌肉中大量肌球蛋白的集体工作行为.利用动力学方程的非平衡定态解,并结合Pate和Cooke的实验结果得到了力作为变量的肌肉态方程.理论结果同热力学原理一致,与传统的肌肉收缩理论有一定区别.根据肌肉的特殊结构,对肌肉态方程做了进一步讨论.     

丝瓜肌球蛋白的电子显微学研究

从丝瓜 (Luffacylindrica (L .)Roem .)卷须中纯化得到分子量为 174kD的肌球蛋白 ,并对其进行了酶学与电子显微学的研究。这种肌球蛋白具有肌动蛋白激活的MgATPase活性 ,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别。电子显微学研究表明 :它有两个头部 (大小和形状与动物肌肉的肌球蛋白相似 )和一条相对较短的尾部。还对丝瓜卷须的肌动蛋白进行了观测 ,偶尔发现一些尾部有球状结构的肌球蛋白。该肌球蛋白的免疫特性和超微结构证明了它由 2条重链组成 ,并与传统的肌球蛋白相似。然而 ,这种 174kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究。     

丝瓜肌球蛋白的电子显微学研究

从丝瓜(Luffa cylindrica (L.) Roem.)卷须中纯化得到分子量为174kD的肌球蛋白,并对其进行了酶学与电子显微学的研究.这种肌球蛋白具有肌动蛋白激活的MgATPase活性,能够被抗动物肌肉的肌球蛋白的单克隆抗体识别.电子显微学研究表明:它有两个头部(大小和形状与动物肌肉的肌球蛋白相似)和一条相对较短的尾部.还对丝瓜卷须的肌动蛋白进行了观测,偶尔发现一些尾部有球状结构的肌球蛋白.该肌球蛋白的免疫特性和超微结构证明了它由2条重链组成,并与传统的肌球蛋白相似.然而,这种174 kD的肌球蛋白是否参与了丝瓜的接触卷曲有待于进一步研究.     

细胞骨架(续)

(四)微丝和微管的功能微丝和微管的功能就三个问题来谈:细胞形状的维持、与非肌肉细胞运动的关系和与细胞分裂的关系。这里要谈到两个运动系统即肌动蛋白—肌球蛋白系统和微管—二联臂(dynein)系统。非肌肉细胞的运动机制,目前多从肌原纤维的收缩蛋白在非肌肉细胞中的分布等方面进行探讨。一旦肌节的单一蛋白鉴定和纯化,就可在非肌肉细胞中寻找是否有相同或近     

人心肌肌球蛋白轻链1与重链和肌动蛋白的结合

在测得中国人心肌肌球蛋白轻链 1cDNA的核苷酸序列 ,并获得一株单克隆抗体 (HCMLC1 8)的基础上 ,用PCR方法 ,以中国人心肌肌球蛋白轻链 1的cDNA为模板 ,分别获得中国人心肌肌球蛋白轻链 1的各为 98个氨基酸的N端和C端片段cDNA的克隆并进行了表达。同时进行了其表达产物和大鼠心肌肌球蛋白重链和人心肌肌动蛋白以及单克隆抗体结合的研究 ,发现三者均和轻链 1的N端相结合 ,结合位点各不相同。这些结合位点可能均位于轻链 1的分子表面 ,而且如果轻链 1在实验状态下先与肌动蛋白结合 ,则有可能影响轻链与重链间的彼此结合。肌动蛋白在体外能以不同位点结合肌球蛋白重链和轻链 ,可能在肌肉收缩过程中具有重要的生理意义     

力蛋白的分子生物学与细胞运动

从亚细胞水平和分子生物学水平研究细胞运动(如:肌肉收缩、纤毛和鞭毛的运动、细胞质流动、染色体的分离、伪足的伸展等等)还是近一、二十年的事。我们已经知道肌肉收缩是由于肌动蛋白(actin)和肌球蛋白(myosin)相互作用的结果,七十年代以来另一种和运动有关的大分子系统越来越引起人们的关注,这就是微管蛋白(tubulin)和力蛋白(dynein~*)系统,现在已经知道纤毛和真核生物鞭毛的运动就是这两种蛋白质相互作用的结果。     

植物细胞内的胞质川流

近二十年来,对真核细胞所表现的各种运动已做了深入的研究,累积了相当数量的知识。非肌肉细胞的运动,目前按照收缩蛋白对,即肌动蛋白——肌球蛋白以及微管蛋白——待宁蛋白来划分。变形虫运动、胞质川流、组织细胞的运动、胞质分裂等属于第一种。纤毛和鞭毛运动、轴杆(axostyles)的弯曲等属于第二种。     

肌肉收缩及自发振动的动力学理论

改变实验条件,肌纤维可发生从收缩到自发振动的相变。为了研究这一现象,引进了描述肌纤维内部弹性成分拉伸长度与张力关系的表达式,利用肌肉态方程并考虑肌纤维的特殊结构,给出了描述肌纤维收缩及自发振动的统一动力学方程。从动力学方程出发,肌纤维自发振动的发生条件得到了自然解释,所给出的振动周期和振动曲线同实验结果相符,并给出了一些新的理论结果。这一工作的意义在于,完成了从肌球蛋白单分子性质、肌纤维组织结构到肌纤维功能的信息整合。     

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.     

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.     

Movement of actin away from the center of reconstituted rabbit myosin filament is slower than in the opposite direction.

By decreasing ionic strength slowly, thick filaments of several micrometers in length were obtained from purified rabbit skeletal muscle myosin. Dark-field observation showed these filaments with their center scattering light extensively. Active movement of actin filaments complexed with tetramethyl rhodamine-phalloidin along the reconstituted myosin filaments was observed. Actin filaments moved towards the center of myosin filaments at a speed of 3.9 +/- 1.6 microns s-1 (mean +/- SD, n = 40) and often continued to move beyond the center towards the tip of the opposite side at a lower speed. The speed of the movement away from the center was 1.0 +/- 0.6 microns s-1 (n = 59). Thus, the functional bipolarity in terms of the movement speed which was first found in native thick filaments of molluscan smooth muscle is also seen in reconstituted filaments from purified rabbit skeletal muscle myosin. The difference of the speed between the two directions is considered to be due to properties of myosin molecules themselves.     

Actin-binding protein amplifies actomyosin contraction,and gelsolin confers calcium control on the direction of contraction

Cross-linking of muscle actin filaments by low concentrations of actin-binding protein reduces the concentration of muscle myosin required for contraction of actin. Gelsolin, a macrophage protein that divides actin filaments in the presence of calcium, inhibits the amplifying effect of actin-binding protein on contraction of actomyosin. In a calcium gradient, the actomyosin gel moves from high to low calcium concentrations, indicating that calcium-controlled lattice formation can impart directionality to the movement of an isotropic actin network.     

An analysis of the possible source of contractile forces in striated muscle

A thermodynamic analysis, based upon current understanding of the molecular structure and chemistry of striated muscle, defines the conditions under which contractile forces may be generated and external work done. Under these conditions tension is generated by crossbridges acting individually and asynchronously, so that tension is proportional to the extent of overlap of actin and myosin filaments. Estimation of the mean isometric tension per crossbridge, in a simple case, gives a value comparable to that observed experimentally. The role of ATP hydrolysis in the generation of tension and performance of work is an indirect one. Isometric tension is maintained at the expense of an enhanced rate of hydrolysis of ATP, and for isotonic contraction the rate of ATP hydrolysis increases with increasing speed of contraction. The mean tension during isotonic contraction varies inversely with the speed of contraction.     

Influence of dispersion in myosin filament orientation and anisotropic filament contractions in smooth muscle

A new constitutive model for the biomechanical behaviour of smooth muscle tissue is proposed. The active muscle contraction is accomplished by the relative sliding between actin and myosin filaments, comprising contractile units in the smooth muscle cells. The orientation of the myosin filaments, and thereby the contractile units, are taken to exhibit a statistical dispersion around a preferred direction. The number of activated cross-bridges between the actin and myosin filaments governs the contractile force generated by the muscle and also the contraction speed. A strain-energy function is used to describe the mechanical behaviour of the smooth muscle tissue. Besides the active contractile apparatus, the mechanical model also incorporates a passive elastic part. The constitutive model was compared to histological and isometric tensile test results for smooth muscle tissue from swine carotid artery. In order to be able to predict the active stress at different muscle lengths, a filament dispersion significantly larger than the one observed experimentally was required. Furthermore, a comparison of the predicted active stress for a case of uniaxially oriented myosin filaments and a case of filaments with a dispersion based on the experimental histological data shows that the difference in generated stress is noticeable but limited. Thus, the results suggest that myosin filament dispersion alone cannot explain the increase in active muscle stress with increasing muscle stretch.     

Vectorial activation of smooth muscle myosin filaments and its modulation by telokin

Smooth muscle myosin copurifies with myosin light chain kinase (MLCK) and calmodulin (CaM) as well as with variable amounts of myosin phosphatase. Therefore, myosin filaments formed in vitro also contain relatively high levels of these enzymes. Thus these filaments may be considered to be native-like because they are similar to those expected to exist in vivo. These endogenous enzymes are present at high concentrations relative to myosin, sufficient for rapid phosphorylation and dephosphorylation of the filaments at rates comparable to those observed for contraction and relaxation in intact muscle strips. The phosphorylation by MLCK/CaM complex appears to exhibit some directionality and is not governed by a random diffusional process. For the mixtures of myosin filaments with and without the endogenous MLCK/CaM complex, the complex preferentially phosphorylates its own parent filament at a higher rate than the neighboring filaments. This selective or vectorial-like activation is lost or absent when myosin filaments are dissolved at high ionic strength. Similar vectorial-like activation is exhibited by the reconstituted filament suspensions, but the soluble systems composed of isolated regulatory light chain or soluble myosin head subfragments exhibit normal diffusional kinetic behavior. At physiological concentrations, kinase related protein (telokin) effectively modulates the activation process by reducing the phosphorylation rate of the filaments without affecting the overall phosphorylation level. This results from telokin-induced liberation of the active MLCK/CaM complex from the filaments, so that the latter can also activate the neighboring filaments via a slower diffusional process. When this complex is bound at insufficient levels, this actually results in acceleration of the initial phosphorylation rates. In short, I suggest that in smooth muscle, telokin plays a chaperone role for myosin and its filaments.     

The mechanism of the movement of leucocytes

In a study of the movement of human leucocytes it was clarified that characteristic contraction waves were observed on the cell surface during movement and an initial morphological change directly related to the appearance of the wave originated in the surface of the granuloplasm and not in the cell membrane. From these findings, together with physicochemical properties of the contractile protein from equine leucocytes, it was proposed that the wave observed in moving leucocytes might be conducted, in some way, by contraction and relaxation of the contractile protein in the cells. Myosin A and actin as constituents of the contractile protein were extracted separately from leucocytes in polymerized form, which resemble myosin aggregate and F-actin from muscle, respectively. The thick and thin filaments of about 150 and 80 Å in diameter were observed in glycerinated leucocytes with electron microscopy. When glycerinated leucocytes were incubated with heavy meromyosin (HMM) from rabbit skeletal myosin A, the thin filaments developed a structure resembling the ‘arrowhead structure’ of the HMM F-actin complex in vitro. The thick filaments seemed to correspond to myosin aggregates and the thin ones to filaments containing F-actin.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.