胞间连丝结构与组分的研究进展

胞间连丝是植物体内连接相邻细胞的一种跨细胞的细胞器,是细胞间物质运输和信息传递的通道,为相邻细胞间小分子、病毒颗粒及一些特殊大分子的运输提供途径。细胞间的共质通道就在质膜和其内部的压缩内质网间形成,压缩内质网又称连丝微管。由于胞间连丝结构的复杂性,使得对其结构组分的研究受到很大限制,但近几年随着电子显微镜、免疫标记、显微注射技术的广泛应用,已有证据表明肌动蛋白、肌球蛋白、中心蛋白和钙网蛋白等是胞间连丝的组分,这些蛋白之间相互作用调节胞间连丝的通透性,控制物质运输。     

Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum

The isolated head fragment of myosin is a motor protein that is able to use energy liberated from the hydrolysis of adenosine triphosphate to cause sliding movement of actin filaments. Expression of a myosin fragment nearly equivalent to the amino-terminal globular head domain, generally referred to as subfragment 1, has been achieved by transforming the eukaryotic organism Dictyostelium discoideum with a plasmid that carries a 2.6-kilobase fragment of the cloned Dictyostelium myosin heavy chain gene under the control of the Dictyostelium actin-15 promoter. The recombinant fragment of the myosin heavy chain was purified 2400-fold from one of the resulting cell lines and was found to be functional by the following criteria: the myosin head fragment copurified with the essential and regulatory myosin light chains, decorated actin filaments, and displayed actin-activated adenosine triphosphatase activity. In addition, motility assays in vitro showed that the recombinant myosin fragment is capable of supporting sliding movement of actin filaments.     

Polo-like kinase Cdc5 controls the local activation of Rho1 to promote cytokinesis

The links between the cell cycle machinery and the cytoskeletal proteins controlling cytokinesis are poorly understood. The small guanine nucleotide triphosphate (GTP)-binding protein RhoA stimulates type II myosin contractility and formin-dependent assembly of the cytokinetic actin contractile ring. We found that budding yeast Polo-like kinase Cdc5 controls the targeting and activation of Rho1 (RhoA) at the division site via Rho1 guanine nucleotide exchange factors. This role of Cdc5 (Polo-like kinase) in regulating Rho1 is likely to be relevant to cytokinesis and asymmetric cell division in other organisms.     

The crystal structure of uncomplexed actin in the ADP state

The dynamics and polarity of actin filaments are controlled by a conformational change coupled to the hydrolysis of adenosine 5'-triphosphate (ATP) by a mechanism that remains to be elucidated. Actin modified to block polymerization was crystallized in the adenosine 5'-diphosphate (ADP) state, and the structure was solved to 1.54 angstrom resolution. Compared with previous ATP-actin structures from complexes with deoxyribonuclease I, profilin, and gelsolin, monomeric ADP-actin is characterized by a marked conformational change in subdomain 2. The successful crystallization of monomeric actin opens the way to future structure determinations of actin complexes with actin-binding proteins such as myosin.     

冷藏条件下鲢鱼肌肉蛋白的变化

利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹法分析比较了鲢鱼在0和4℃下冷藏14 d过程中肌肉蛋白的变化情况.结果表明:在2种温度下冷藏,肌浆蛋白降解均不显著,其变化趋势基本一致;肌球蛋白重链在0℃下冷藏14 d,4℃下冷藏11 d后发生明显降解;α-辅肌动蛋白和肌动蛋白在2种温度下冷藏均呈逐渐降解的趋势,且在4℃下冷藏降解更为显著;原肌球蛋白在0和4℃下冷藏2 d后均开始降解,此后于0℃冷藏蛋白无明显变化,但于4℃冷藏11 d后降解速度加快.     

蛋白质磷酸化对肌动球蛋白解离及其乙酰化水平的影响

[目的]研究肌球蛋白重链和肌动蛋白磷酸化对其乙酰化水平、肌动球蛋白解离及ATP酶活性的影响,为通过调控磷酸化水平改善肉品嫩度提供理论依据.[方法]以羊背最长肌为材料制备肌肉匀浆液,采用碱性磷酸酶抑制剂(抑制去磷酸化)和蛋白激酶抑制剂(抑制磷酸化)调控其磷酸化水平,在4℃分别孵育0、0.5、4、12、24、48和72 h...     

Polarity and velocity of sliding filaments: control of direction by actin and of speed by myosin

Myosin filaments, which are responsible for a large repertoire of motile activities in muscle and nonmuscle cells, can translocate actin filaments both toward and away from their central bare zone. This bidirectional movement suggests that there is enough flexibility in the head portion of the tightly packed myosin molecules in the native myosin filaments to move actin filaments not only in the expected direction, but also in the direction opposite to that predicted by the regular structure of muscle--away from the center of the myosin filament.     

Muscle contraction and free energy transduction in biological systems

Muscle contraction occurs when the actin and myosin filaments in muscle are driven past each other by a cyclic interaction of adenosine triphosphate (ATP) and actin with cross-bridges that extend from myosin. Current biochemical studies suggest that, during each adenosine triphosphatase cycle, the myosin cross-bridge alternates between two main conformations, which differ markedly in their strength of binding to actin and in their overall structure. Binding of ATP to the cross-bridge induces the weak-binding conformation, whereas inorganic phosphate release returns the cross-bridge to the strong-binding conformation. This cross-bridge cycle is similar to the kinetic cycle that drives active transport and illustrates the general principles of free energy transduction by adenosine triphosphatase systems.     

Calcium-sensitive cross-bridge transitions in mammalian fast and slow skeletal muscle fibers

The fundamental mechanism underlying the differing rates of tension development in fast and slow mammalian skeletal muscle is still unknown. Now, in skinned (membrane-permeabilized) single fibers it has been shown that the rate of formation of the strongly bound, force-producing cross-bridge between actin and myosin is calcium-sensitive in both fast and slow fibers and that the rate is markedly greater in fast fibers. The transition rates obtained at high calcium concentrations correlated with myosin isoform content, whereas at low calcium concentrations the thin filament regulatory proteins appeared to modulate the rate of tension development, especially in fast fibers. Fiber type-dependent differences in rates of cross-bridge transitions may account for the characteristic rates of tension development in mammalian fast and slow skeletal muscles.     

光对美洲蛙肌肉纤维衍射强度的分布研究

肌原纤维是由粗丝和细丝重迭而成的Ａ带和只含细丝的Ⅰ带组成，形成了天然光栅，因此可用光学方法探讨肌原纤维分子结构及其动力学问题。试验表明，单色光通过美洲蛙肌原纤维后，衍射光左右两端为非对称性，且左右条纹锋值间隔随肌原纤维节长度增大而减小。肌肉运动有张有弛，于是肌原纤维长度有变异，共变异与非对称有关。当肌原纤维长度增加时，左右两端强度差异变大，而相对应条纹的锋值间隔距离变小。这一现象与布拉格方程和折射     

Actin-myosin interaction: inhibition of the myosin adenosine triphosphatase by actin

In the absence of magnesium ion, the addition of actin to myosin in a 1 :4 ratio has a strong inhibitory effect on the adenosine triphosphatase activity, in contrast to the well-known activating effect of actin in the presence of magnesium ion. This finding suggests that both effects result from a conformational change in the active site of the myosin adenosine triphosphatase.     

Assembly mechanism of the contractile ring for cytokinesis by fission yeast

Animals and fungi assemble a contractile ring of actin filaments and the motor protein myosin to separate into individual daughter cells during cytokinesis. We used fluorescence microscopy of live fission yeast cells to observe that membrane-bound nodes containing myosin were broadly distributed around the cell equator and assembled into a contractile ring through stochastic motions, after a meshwork of dynamic actin filaments appeared. Analysis of node motions and numerical simulations supported a mechanism whereby transient connections are established when myosins in one node capture and exert force on actin filaments growing from other nodes.     

宰后肌肉中肌球蛋白磷酸化调控肌动球蛋白解离作用机制

【目的】研究宰后肌肉中肌球蛋白磷酸化与肌动球蛋白解离之间的关系,分析其磷酸化水平的变化对肌动球蛋白解离的影响,探究肌球蛋白磷酸化对宰后肌肉肌节长度与嫩度的作用。【方法】取宰后30 min内的羊背最长肌,在4℃条件下分别成熟6、24、48和72 h,通过SDS-PAGE电泳、Pro-Q染色和蛋白质免疫印迹测定肌球蛋白的磷酸化水平和肌动球蛋白解离程度随宰后时间的变化;测定肌动球蛋白ATP酶的活性,分析宰后不同时间点肌球蛋白与肌动蛋白结合作用力的强弱;采用透射电镜分析宰后肌节长度随时间的变化。【结果】研究发现宰后肌肉中肌球蛋白轻链2的磷酸化水平在0.5—48 h快速降低(P0.05),并在48 h达到最低点,在48—72 h有所升高(P0.05),但其最终磷酸化水平明显低于初始值。肌动球蛋白的解离程度在宰后初期(0.5—6 h)显著降低(P0.05),在6—48 h显著升高(P0.05),并于48—72 h维持稳定,其最终解离程度显著高于宰后0.5 h的初始值。肌动球蛋白ATPase活性在宰后初期(0.5—6 h)略有升高,6—24 h快速上升(P0.05),并在24 h达到最高点,24—72 h逐渐降低;而肌节长度的变化则与之相反,呈先下降后上升的趋势,并在24 h达到肌节最短点。【结论】羊宰后肌肉中的肌球蛋白轻链2磷酸化水平的变化对肌球蛋白与肌动蛋白的相互作用有较大的影响,且肌节收缩(肌球蛋白与肌动蛋白的相互作用力)与肌动球蛋白的解离(肌球蛋白与肌动蛋白的相互作用量)并不是一个同步的进程。肌球蛋白轻链2的磷酸化修饰负向调控肌动球蛋白解离和肌动球蛋白ATPase活性,导致肌节的收缩与舒张,进而调控肉品最终的嫩度。     

Actin network architecture can determine myosin motor activity

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.     

Nonequilibrium mechanics of active cytoskeletal networks

Cells both actively generate and sensitively react to forces through their mechanical framework, the cytoskeleton, which is a nonequilibrium composite material including polymers and motor proteins. We measured the dynamics and mechanical properties of a simple three-component model system consisting of myosin II, actin filaments, and cross-linkers. In this system, stresses arising from motor activity controlled the cytoskeletal network mechanics, increasing stiffness by a factor of nearly 100 and qualitatively changing the viscoelastic response of the network in an adenosine triphosphate-dependent manner. We present a quantitative theoretical model connecting the large-scale properties of this active gel to molecular force generation.     

Microinjected DNA from the X chromosome affects sex determination in Caenorhabditis elegans

The signal for sex determination in the nematode Caenorhabditis elegans is the ratio of the number of X chromosomes to the number of sets of autosomes (X/A ratio). By previous genetic tests, elements that feminized chromosomal males appeared to be widespread on the X chromosome, but the nature of these elements was not determined. In experiments to define a feminizing element molecularly, cloned sequences were added to chromosomally male embryos by microinjection into the mother. Three different X-chromosome clones, including part of an actin gene, part of a myosin heavy chain gene, and all of two myosin light chain genes, feminize chromosomal males. Both somatic and germline aspects of sex determination are affected. In contrast, about 40 kilobases of nematode autosomal DNA, phage lambda DNA, and plasmid pBR322 DNA do not affect sex determination. A feminizing region was localized to a maximum of 131 base pairs within an intron of the X-linked actin gene; a part of the gene that does not have this region is not feminizing. The results suggest that short, discrete elements found associated with many X-linked genes may act as signals for sex determination in C. elegans.     

Electron microscopic evidence for plasmodesmata in dicotyledonous guard cells

In Nicotianna tobaccum and Vicia faba leaves, plasmodesmata were observed by electron microscopy in walls between sister guard cells and walls between guard and epidermal cells. The latter were found primarily in pit fields of anticlinal walls and showed considerable complexity as evidenced by branching. Cytologically, the plasmodesmata appear functional in operative guard cells and should be considered in the mechanism of stomatal movements.     

Laser-stimulated luminescence used to measure x-ray diffraction of a contracting striated muscle

An integrating x-ray area detector that operates on the basis of laser-stimulated luminescence was used in a diffraction study of muscle contraction. The area detector has a dynamic range of 1 to 10(5), a sensitivity about 60 times greater with approximately 1/300 as much fog background as x-ray film. It is erasable and reusable but, like film, can integrate at a practically unlimited counting rate. The high sensitivity and wide dynamic range of the detector resulted in a sufficient reduction in the exposure time to make possible the recording of a clear x-ray diffraction pattern, with up to 2.0-nanometer axial spacing, from a contracting frog skeletal muscle in as little as 10 seconds with synchrotron radiation. During the isometric contraction of the muscle, most of the actin diffraction lines increased in intensity without noticeable changes in their peak positions. Changes also occurred in diffraction intensities from the myosin heads. The results indicate that during contraction the structure of the actin filaments differs from that in the rigor state, suggesting a possible structural change in the actin subunits themselves; the myosin heads during contraction retain the axial periodicity of the myosin filament and become aligned in a more perpendicular manner to the actin filaments.     

蛋白质磷酸化调控羊肉肌原纤维蛋白的功能

【目的】通过激活蛋白激酶的活性提高羊肉肌原纤维蛋白的磷酸化水平,分析磷酸化水平的提高对羊肉肌原纤维蛋白降解程度、收缩功能的影响,进一步揭示蛋白质磷酸化对羊肉肌肉嫩化的作用机理。【方法】通过添加蛋白激酶A激活剂Forskolin、蛋白激酶C激活剂佛波酯（PMA）,提高蛋白激酶的活性,改变羊肉肌原纤维蛋白的磷酸化水平。比较激活剂处理组与空白对照组肌原纤维小片化指数（MFI）、条带降解程度、肌节长度等指标的差异,确定蛋白质磷酸化水平对羊肉肌肉收缩和肌肉降解的影响。【结果】将羊肉样品在蛋白激酶溶液中培养24 h,在培养结束后1、2和4 h,PMA处理组（PKC激活组）的蛋白激酶活性显著高于空白对照组（P＜0.05）,而在培养后1和4 h,Forskolin处理组（PKA激活组）的蛋白激酶活性显著高于空白对照组（P＜0.05）。PMA处理组和Forskolin处理组的最高蛋白激酶活性出现在培养后1 h。Forskolin和PMA通过提高激酶活性显著提高肌联蛋白（Titin）、肌球蛋白结合蛋白C（Myosin binding protein C）、原肌球蛋白（Tropomyosin）、肌球蛋白轻链2（Myosin light chain 2）等蛋白质的磷酸化水平,肌球蛋白重链、肌动蛋白质的磷酸化水平没有显著变化,Forskolin组和PMA组的蛋白质降解程度及肌节长度均低于对照组。【结论】肌原纤维蛋白磷酸化水平提高不利于羊肉肌原纤维蛋白的降解。另一方面,磷酸化水平可能通过对肌球蛋白轻链2的作用增强羊肉肌肉的收缩作用力,通过促进相邻原肌球蛋白的相互作用促进肌细丝收缩,影响肉的僵直进程和嫩化进程。     

生物分子马达动力冲程研究

通过建立肌球蛋白工作循环的四态模型,利用化学动力学方法和生化热力学原理,计算了动力冲程的大小不仅依赖于动力冲程过程的反应平衡常数及动力冲程前态的分离速率常数,还与肌球蛋白横桥的弹性系数有关。同时,讨论了肌球蛋白横桥的弹性系数与力的关系,为试验研究提供理论依据。