The dance of actin and myosin: a structural and spectroscopic perspective

Actin and myosin interact in a cyclic series of steps linked to the hydrolysis of ATP that are representative of an ancient and widespread molecular mechanism. Spectroscopic findings are related to the analysis of the actin and myosin structures and results from kinetics, fibers, single molecules, electron microscopy, genetics, and a variety of other biophysical and biochemical studies on actin and myosin to provide an overview of the steps in this molecular process. The synthesis of the key findings from these fields reveals a highly efficient engine that amplifies subtle changes in the active site into unsurpassed molecular displacements. Recent developments in resonance energy-transfer spectroscopy and X-ray crystallography are enabling a detailed elucidation of the stages of a large power stroke that concurs with evidences from diverse lines of structural and kinetic inquiry. A complete image of actin and myosin motility appears to include twists, tilts, steps, and dynamics from both partners that could be described as a molecular dance.     

Myosins and pathology: genetics and biology

This article summarizes current knowledge on the genetics and possible molecular mechanisms of Human pathologies resulted from mutations within the genes encoding several myosin isoforms. Mutations within the genes encoding some myosin isoforms have been found to be responsible for blindness (myosins III and VIIA), deafness (myosins I, IIA, IIIA, VI, VIIA and XV) and familial hypertrophic cardiomyopathy (beta cardiac myosin heavy chain and both the regulatory and essential light chains). Myosin III localizes predominantly to photoreceptor cells and is proved to be engaged in the vision process in Drosophila. In the inner ear, myosin I is postulated to play a role as an adaptive motor in the tip links of stereocilia of hair cells, myosin IIA seems to be responsible for stabilizing the contacts between adjacent inner ear hair cells, myosin VI plays a role as an intracellular motor transporting membrane structures within the hair cells while myosin VIIA most probably participates in forming links between neighbouring stereocilia and myosin XV probably stabilizes the stereocilia structure. About 30% of patients with familial hypertrophic cardiomyopathy have mutations within the genes encoding the beta cardiac myosin heavy chain and both light chains that are grouped within the regions of myosin head crucial for its functions. The alterations lead to the destabilization of sarcomeres and to a decrease of the myosin ATPase activity and its ability to move actin filaments.     

Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease

The kinesin, dynein, and myosin superfamily molecular motors have fundamental roles in neuronal function, plasticity, morphogenesis, and survival by transporting cargos such as synaptic vesicle precursors, neurotransmitter and neurotrophic factor receptors, and mRNAs within axons, dendrites, and synapses. Recent studies have begun to clarify the mechanisms of cargo selection and directional transport in subcellular compartments. Furthermore, molecular genetics has revealed unexpected roles for molecular motors in brain wiring, neuronal survival, neuronal plasticity, higher brain function, and control of central nervous system and peripheral nervous system development. Finally, it is also evident that molecular motors are critically involved in neuronal disease pathogenesis. Thus, molecular motor research is becoming an exciting frontier of neuroscience.     

Characterization of the minimum domain required for targeting budding yeast myosin II to the site of cell division

Background  

All eukaryotes with the exception of plants use an actomyosin ring to generate a constriction force at the site of cell division (cleavage furrow) during mitosis and meiosis. The structure and filament forming abilities located in the C-terminal or tail region of one of the main components, myosin II, are important for localising the molecule to the contractile ring (CR) during cytokinesis. However, it remains poorly understood how myosin II is recruited to the site of cell division and how this recruitment relates to myosin filament assembly. Significant conservation between species of the components involved in cytokinesis, including those of the CR, allows the use of easily genetically manipulated organisms, such as budding yeast (Saccharomyces cerevisiae), in the study of cytokinesis. Budding yeast has a single myosin II protein, named Myo1. Unlike most other class II myosins, the tail of Myo1 has an irregular coiled coil. In this report we use molecular genetics, biochemistry and live cell imaging to characterize the minimum localisation domain (MLD) of budding yeast Myo1.     

肌球蛋白工作循环的机械化学偶联模型

目前在众多的分子马达中对骨骼肌肌球蛋白的研究较多,本文对肌球蛋白的结构、工作循环机制以及单分子动力学性质进行了探索。同时,对各种生化条件下肌纤维的收缩性质进行了测试。将Houdusse和Sweeney给出的机械化学偶联模型简化成一个新的四态模型,通过对定态时肌球蛋白态分布的研究,证明了简化模型的合理性。     

Purification and properties of myosin from the "hatching muscle" (m. complexus) of geese

The present work is concerned with the study of myosin fractions prepared from the hatching muscle (m. complexus) and a control muscle (m. pectoralis) of the developing goose embryo. The m. complexus attained its maximum mass at hatching and in the 4-day-old bird the mass of this muscle was only one fourth of that recorded at hatching. The m. complexus was hypertrophied already on the 21st day. At days 21, 27 and 28 of incubation and at posthatching days myosin preparations were made from both muscles. Partial purification of myosins from both sources yielded a high molecular weight fraction characteristic of the adult bird and one other protein fraction with molecular mass half of myosin. Both preparations exhibited the characteristic properties of myosin. The lower molecular weight fraction was also shown to develop filamentous aggregates as did the higher molecular-weight, gel filtrated myosin. The phosphate content of the half molecular mass myosin fraction prepared from the embryonic m. complexus at days prior to hatching was considerably higher than that of the high molecular weight fraction and the predominant component was P-Arg. Since the embryonic myosin was still not available in the m. complexus of the 4-day-old birds and the hypertrophied muscle underwent regression after hatching it appears that this myosin fraction is actively involved in breaking through the shell during the hatching period in geese.     

Two homogeneous myosins in body-wall muscle of Caenorhabditis elegans

Myosin purified from the body-wall muscle-defective mutant E675 of the nematode. Caenorhabditis elegans, has heavy chain polypeptides which can be distinguished on the basis of molecular weight. On SDS-polyacrylamide gels, bands are found at 210,000 and 203,000 daltons. This is in contrast to myosin from the wild-type, N2, which has a single heavy chain band at 210,000 daltons. Both heavy chains of E675 are found in body-wall muscle (Epstein, Waterston and Brenner, 1974).When native myosin from E675 is fractionated on hydroxyapatite, it is separated into myosin containing predominantly one or the other molecular weight heavy chain and myosin containing a mixture of the heavy chains. Comparison of the CNBr fragments of myosin that contains predominantly 210,000 dalton heavy chains with those of myosin that contains predominantly 203,000 dalton heavy chains reveals multiple differences. These differences are not explained by the difference in molecular weight of the heavy chains, but may be explained if each type of heavy chain is the product of a different structural gene. Furthermore, because there are fractions which exhibit >80% 210,000 or >80% 203,000 dalton heavy chain, there is myosin which is homogeneous for each of the heavy chains.Although N2 myosin has only a single molecular weight heavy chain, it too is fractionated by hydroxyapatite. By comparing the CNBr fragments of different myosin fractions, we show that N2, like E675, has two kinds of heavy chains.E190, a body-wall muscle-defective mutant in the same complementation group as E675, is lacking the myosin heavy chain affected by the e675 mutation. This property has allowed us to determine by co-purification of labeled E190 myosin in the presence of excess, unlabeled E675 myosin that most, if not all, of the myosin that contains two different molecular weight heavy chains is due to the formation of complexes between homogeneous myosins and not to a heterogeneous myosin.     

Changes in myosin and myosin light chain kinase during myogenesis

Myosins and myosin light chain kinases have been isolated from a cloned line of myoblasts (L5/A10) as this cell line undergoes differentiation toward adult muscle. At least three myosin isozymes were obtained during this developmental process. Initially a nonmuscle type of myosin was found in the myoblasts. The molecular weights of the myoblast light chains were 20 000 and 15 000. Myosin isolated from early myotubes had light chains with molecular weights of 20 000 and 19 500. Myosin isolated from myotubes which contained sarcomeres had light chains with molecular weights of 23 000, 18 500, and 16 000. This last myosin was similar in light chain complement to adult rat thigh muscle. Two forms of the myosin light chain kinase activity were detected: a calcium-independent kinase in the myoblasts and a calcium-dependent kinase in the myotubes with sarcomeres. No myosin light chain kinase activity was detected in the early myotubes.     

Chick brain actin and myosin. Isolation and characterization

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.     

Essential Light Chains of Myosin and Their Role in Functioning of the Myosin Motor

This review summarizes current data on the structure and functions of myosin essential light chains (ELCs) and on their role in functioning of the myosin head as a molecular motor. The data on structural and functional features of the N-terminal extension of myosin ELC from skeletal and cardiac muscles are analyzed; the role of this extension in the ATP- dependent interaction of myosin heads with actin in the molecular mechanism of muscle contraction is discussed. The data on possible interactions of the ELC N-terminal extension with the myosin head motor domain in the myosin ATPase cycle are presented, including the results of the authors’ studies that are in favor of such interactions.     

Cytoplasmic myosin from Drosophila melanogaster

Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized.     

Actin and Myosin in pea tendrils

We demonstrate here the presence of actin and myosin in pea (Pisum sativum L.) tendrils. The molecular weight of tendril actin is 43,000, the same as rabbit skeletal muscle actin. The native molecular weight of tendril myosin is about 440,000. Tendril myosin is composed of two heavy chains of molecular weight approximately 165,000 and four (two pairs) light chains of 17,000 and 15,000. At high ionic strength, the ATPase activity of pea tendril myosin is activated by K⁺-EDTA and Ca²⁺ and is inhibited by Mg²⁺. At low ionic strength, the Mg²⁺-ATPase activity of pea tendril myosin is activated by rabbit skeletal muscle F-actin. Superprecipitation occurred after incubation at room temperature when ATP was added to the crude actomyosin extract. It is suggested that the interaction of actin and myosin may play a role in the coiling movement of pea tendril.     

Myosin from abdominal flexor muscle in a crayfish, Procambarus clarki Girard.

1. Crayfish (Procambarus clarki) myosin was obtained from abdominal flexor muscle. The Ca2+-ATPase activity of crayfish myosin was much lower than that of rabbit skeletal myosin. However, F-actin-activated Mg2+-ATPase of crayfish and its superprecipitation closely resembled those of rabbit skeletal myosin. This fact suggests that the ability of crayfish myosin to combine with F-actin is essentially the same as that of skeletal myosin, although the chemical structures of both the myosin molecules when involved in their Ca2+-ATPast activity must be different from each other. 2. Crayfish and rabbit skeletal myosins were subjected to SDS-polyacrylamide gel electrophoresis. Crayfish myosin was found to have one heavy chain and two distinct light chain components (CF-gl and CF-g2), which have molecular weights of 18,000 and 16,000, respectively. These light chains correspond in molecular weight to the light chains (SK-g2 and SK-g3) in rabbit skeletal myosin. 3. CF-g1 could be liberated from the crayfish myosin molecule reacting with 5,5'-dithio-bis (2-nitrobenzoic acid), (Nbs2), without recovery of ATPase activity by the addition of DTT. These properties are equivalent to those of SK-g2 in rabbit skeletal myosin, although Nbs2-treated crayfish myosin did not recover its ATPase activity at all.     

Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world

In characean algae, very rapid cytoplasmic streaming is generated by sliding movement of an unconventional myosin on fixed actin cables. The speed of this sliding movement is the fastest among many molecular motors known so far. We have cloned a set of overlapping cDNAs encoding the heavy chain of this myosin by immunoscreening with antibody raised against characean myosin. The molecular mass of this heavy chain is 248 kDa, and the protein has a conserved motor domain, six IQ motifs, an extensive alpha-helical coiled-coil domain, and a C-terminal globular domain. Phylogenetic analysis suggested that this myosin belongs to class XI.     

Studies on the subunits of myosin from muscle layer of Ascaris lumbricoides suum.

1. A purified preparation of Ascaris myosin was obtained from the muscle layer of Ascaris lumbricoides suum, using gel filtration and ion-exchange chromatography. 2. Ascaris myosin whether purified or unpurified, had almost the same ability for ATP-splitting and superprecipitation. 3. Ascaris myosin and rabbit skeletal myosin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A significant difference in the number of light chains between both myosins was found. Ascaris myosin was found to have one heavy chain and two distinct light chain components (LC1-A and LC2-A), having molecular weights of 18000 and 16000, respectively. These light chains correspond in molecular weight to the light chain 2 (LC2-S) and light chain 3 (LC3-S) in rabbit skeletal myosin. 4. LC1-A could be liberated from the Ascaris myosin molecule reacted with 5,5'-dithio-bis(2-nirobenzoic acid( Nbs2) with recovery of ATPase activity by addition of dithiothreitol. These properties are equivalent to those of the LC2-S in rabbit skeletal myosin, although Ascaris myosin when treated with Nbs2-urea lost its ATPase activity.     

Selective removal of the carboxyl-terminal tail end of the Dictyostelium myosin II heavy chain by chymotrypsin

Dictyostelium myosin II is a conventional myosin consisting of two heavy chains of 243,000 Da and two pairs of light chains of 16,000 and 18,000 Da. In this paper, we show that the heavy chain of myosin II can be rapidly and selectively cleaved by chymotrypsin to yield two fragments with molecular weights of 195,000 and 38,000 Da as estimated from sodium dodecyl sulfate-polyacrylamide gels. Chymotryptic cleavage at this site occurs most readily in the absence of salt and is greatly inhibited as the salt concentration is increased from 0 to 60 mM. Amino acid sequence analysis of the small fragment demonstrates that its amino terminus corresponds to lysine 1826 of the myosin II heavy chain. If the fragment extends to the carboxyl terminus of the myosin II heavy chain, it would have a molecular weight of 33,700. Upon digestion of myosin II which has been phosphorylated with a high molecular weight Dictyostelium myosin heavy chain kinase (C?té, G.P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072), all of the phosphate is recovered on the 33,700-Da tail-end fragment. Chymotrypsin-cleaved myosin II is shown to be capable of forming filaments at salt concentrations between 20 and 100 mM as judged by its ability to be sedimented by centrifugation. Only the large fragment of myosin II is found in the pellet; the 33,700-dalton fragment remains soluble. Chymotrypsin-cleaved myosin II can bind to actin and displays a high Ca2+-activated ATPase activity but has very low actin-activated ATPase activity.     

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

植物分子群体遗传学研究动态

分子群体遗传学是当代进化生物学研究的支柱学科, 也是遗传育种和关于遗传关联作图和连锁分析的基础理论学科。分子群体遗传学是在经典群体遗传的基础上发展起来的, 它利用大分子主要是DNA序列的变异式样来研究群体的遗传结构及引起群体遗传变化的因素与群体遗传结构的关系, 从而使得遗传学家能够从数量上精确地推知群体的进化演变, 不仅克服了经典的群体遗传学通常只能研究群体遗传结构短期变化的局限性, 而且可检验以往关于长期进化或遗传系统稳定性推论的可靠程度。同时, 对群体中分子序列变异式样的研究也使人们开始重新审视达尔文的以“自然选择”为核心的进化学说。到目前为止, 分子群体遗传学已经取得长足的发展, 阐明了许多重要的科学问题, 如一些重要农作物的DNA多态性式样、连锁不平衡水平及其影响因素、种群的变迁历史、基因进化的遗传学动力等, 更为重要的是, 在分子群体遗传学基础上建立起来的新兴的学科如分子系统地理学等也得到了迅速的发展。文中综述了植物分子群体遗传研究的内容及最新成果。     

Biological ion exchanger resins: XI. Actin in Escherichia coli.

The 45,000 molecular weight component of an "actin-like" fraction (A-L fraction) from E. coli is identified as actin. Passage of skeletal muscle myosin through the A-L fraction specifically removes the 45,000 molecular weight band visible on electrophoresis prior to passage. Examination of the myosin after passage exhibits a new band on electrophoresis at 45,000 molecular weight.     

Qualitative analysis of skeletal myosin as substrate of Ca2+-activated neutral protease: comparison of filamentous and soluble, native, and L2- deficient myosin

Ca2+ -activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions where native myosin was the substrate, CAF selected for the "head" region of the myosin heavy chain: (a) large heavy chain fragments of identical molecular weight were produced from filamentous and from soluble myosin; (b) light meromyosin was not a substrate; (c) agents known to bind to the head of myosin (actin, MgATP, and L2) had both a qualitative and quantitative effect on degradation; and (d) similar cleavage sites could be demonstrated for myosin and for heavy meromyosin (HMM) despite the fact that HMM was a much poorer substrate than myosin. This observation is interpreted as an indication that the conformation of myosin heavy chain is altered in the preparation of HMM. The principal cleavage sites on the heavy chain of myosin were 20,000, 35,000 and 50,000 D from the N-terminus, producing large fragments with molecular weights of 180,000, 165,000, and 150,000 which comprised a "nicked" species of myosin. This nicked species retained both normal solubility properties and normal hydrolytic activities. For this reason, it is concluded that "nicked myosin" is an important pathophysiological species.