植物微管结合蛋白AtMAP65-1在拟南芥气孔保卫细胞中的定位(简报)

植物细胞微管骨架的不同排列方式对细胞的生长分化及形态建成具有重要意义,微管的这种动态组织行为不仅需要自身的组成蛋白-微管蛋白(tubulin),还要有微管辅助蛋白MAPs(Microtubule-associated proteins)的参与[1,2]。即MAPs是一类能够与微管骨架特异结合并调节其动态装配过程及其结构、进而影响微管功能的蛋白大分子。其中,MAP65是最先在烟草悬浮细胞BY-2中纯化出来的、分子量约为65KDa的一个微管结合蛋白家族。     

靶向微管抗肿瘤药物研究进展

微管由微管蛋白组成,在细胞分裂、细胞内物质运输、信号传递、维持细胞形态等过程中起着重要作用.一些干扰微管功能的化合物可使细胞停滞在有丝分裂期而抑制细胞增殖.相对于正常细胞,肿瘤细胞有丝分裂异常频繁,以微管作为抗肿瘤的靶点已成为研究热点.作用于微管的微管蛋白抑制剂通过抑制微管蛋白的聚合促进微管解聚或者抑制微管解聚促进微管蛋白聚合来破坏微管动态平衡、干扰肿瘤细胞纺锤体形成、阻断细胞分裂、抑制肿瘤增殖,现就微管蛋白抑制剂的研究进展作一综述.     

激素引起伊贝母愈伤组织微管蛋白变化的免疫细胞化学研究(简报)

微管蛋白聚合形成微管。微管在维持细胞结构、物质运输、分裂及植物细胞壁的建成等过程中起着重要的作用。70年代后期,在微管生物化学研究取得很大进展的基础上,免疫细胞化学技术与微管研究结合起来,使人们能够从整体水平观察以微管蛋白为主要成份的细胞骨架的动态变化。我们采用免疫酶标技术,对生长在含不同激素培养基上的伊贝母愈伤组织的微管及微管蛋白变化进行了观察和分析,结果表明,激素种类和微管的存在形式是相关的。     

玉米α-微管蛋白分子生物学研究进展

自１９６３ 年在植物细胞中发现微管以来,其研究取得了较大进展。α＿ 微管蛋白是组成微管的基本单位之一。本文综述了玉米α＿微管蛋白基因及其表达调控的研究进展     

玉米a-微管蛋白分子生物学研究进展

自1963年在植物细胞中发现微管以来,其研究取得了较大进展。α-微管蛋白是组成微管的基本单位之一。本文综述了玉米α-微管蛋白基因及其表达调控的研究进展。     

植物微管骨架参与下胚轴伸长调节机制研究进展

微管作为细胞骨架的重要成员,在植物生长发育过程中起重要作用。下胚轴作为研究细胞伸长的模式系统之一,其伸长受到多种信号的调节。该文综述了微管骨架在响应环境和生长发育信号调节下胚轴伸长过程中的作用及机制,旨在帮助读者深入理解微管骨架响应上游信号在植物下胚轴伸长中的作用机理。     

微管成核的研究进展

微管成核是指微管蛋白(tubulin)分子相互作用形成微管组织“核心”的过程,它是微管形成的初始阶段。在一定条件下,微管蛋白溶液中可以发生微管成核现象。γ微管蛋白(γ-tubulin)或多种γ微管蛋白复合体的存在能够加速这一过程。在体内,一般是由γ-TuRC(γ-tubulin ring complex)启动微管的装配。近年来研究发现即使没有γ微管蛋白,机体仍然能够利用某种机制组织微管成核。     

秋水仙碱对微管蛋白的作用机制及其细胞效应研究进展

秋水仙碱诱导染色体同源加倍的生物学机理与构成纺锤体微管蛋白密切相关. 秋水仙碱作用于细胞的根本效应是改变细胞微管的状态,使微管解聚或停止组装;秋水仙碱作用于微管的方式是其分子结构中的 A 环与β微管蛋白354半胱氨酸结合、C环结合在239半胱氨酸和N末端氨基酸;不同植物种类微管蛋白的处理效应有明显的差异.秋水仙碱的处理效应影响到细胞一切与微管活动有关的功能,具体表现为改变细胞的发育进程、阻断染色体的分裂及细胞器不能正常运动,除此之外,秋水仙碱还可以诱导染色体结构变异.本文主要综述了秋水仙碱作用于微管蛋白的机制及秋水仙碱处理的细胞效应等研究进展,为该领域的研究提供信息资料.     

植物微管和微丝骨架研究的进展

从以上叙述的资料中可以看出,近年来在植物微管蛋白的分离及其化学性质、微管的组织中心、微管的异质性、微丝的分布,以及微管和微丝骨架的功能及基因调节等方面的研究取得不少新的进展;特别是从植物中直接分离微管蛋白取得成功、以及微管蛋白异型、微管冷稳定性与植物抗寒性的关系及微丝分布广泛性等的发现,对植物细胞骨架的进一步研究具有重要意义。     

绿豆根尖细胞微管骨架有丝分裂时相发育变化的研究

提纯猪脑微管蛋白,制备兔抗微管蛋白抗血清,以此抗体与羊抗兔ｌｇＧ－ＦＩＴＣ因清,对绿豆根尖细胞进行间接免疫荧光标记和荧光显微镜检,得到了绿豆根尖细胞有丝分裂微管骨架周期发育变化的时相,如：早前期带,纺棰体微管,成膜体微管等,结果证明了双子叶植物具有与单子叶植物相似的细胞分裂微管周期时相,表明了微管架周期时相变化在高等植物中具有普遍性和共同变化的规律,讨论了微管骨架时相发育变化与染色有丝分裂行为的关     

Three-dimensional reconstruction of tubulin in zinc-induced sheets: II. Consequences of removal of microtubule associated proteins

The three-dimensional structure of zinc-induced tubulin sheets freed of microtubule associated proteins has been determined to 20 Å resolution by electron microscopy and image reconstruction. The determination was carried out with porcine brain tubulin separated from microtubule associated proteins by phosphocellulose chromatography. Negatively stained samples were tilted using the goniometer stage of the electron microscope to provide images of the tubulin sheets ranging in tilt from ?60 ° to +60 °. The micrographs were digitized and subjected to a cross-correlation analysis to compensate for smooth curvature of the lattice in the sheets. For each angle of tilt, an average unit cell was obtained from the cross-correlation analysis and subsequently a Fourier transform was computed for inclusion in the three-dimensional Fourier data set. The transforms of 47 tilted images plus the average of five untilted sheets were combined and an inverse Fourier transform was applied to give a threedimensional reconstruction of the microtubule associated protein-free tubulin sheets. Comparison of the protofilament structure in these sheets with the previously published protofilament structure of zinc-induced tubulin sheets containing microtubule associated proteins reveals a number of consequences of the removal of microtubule associated proteins. (1) The extensive internal contact along the protofilament observed in microtubule associated protein-containing tubulin sheets is maintained in microtubule associated protein-free tubulin sheets. (2) In projection, the protofilaments in microtubule associated protein-free tubulin sheets are 2.2 Å closer together than in microtubule associated protein-tubulin sheets. (3) The deviations of adjacent protofilaments from the plane of the sheets when viewed end-on are more pronounced in the absence of microtubule associated proteins. Differences are also observed at the level of individual tubulin subunits. In particular, the distinct cleft which was found in one class of subunits in tubulin sheets with microtubule associated proteins is absent in the microtubule associated protein-free tubulin sheets. The loss of this cleft and some changes in the shape of the tubulin subunits upon removal of microtubule associated proteins suggest a possible site for the interaction of tubulin with microtubule associated proteins.     

Microtubule nucleation from stable tubulin oligomers

Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed of 10-15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by gamma-tubulin ring complexes with the exception that in the absence of gamma-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.     

Direct interaction of Bcl-2 proteins with tubulin

A direct interaction between tubulin and several pro-apoptotic and anti-apoptotic members of the Bcl-2 family has been demonstrated by effects on the assembly of microtubules from pure rat brain tubulin. Bcl-2, Bid, and Bad inhibit assembly sub-stoichiometrically, whereas peptides from Bak and Bax promote tubulin polymerization at near stoichiometric concentrations. These opposite effects on microtubule assembly are mutually antagonistic. The BH3 homology domains, common to all members of the family, are involved in the interaction with tubulin but do not themselves affect polymerization. Pelleting experiments with paclitaxel-stabilized microtubules show that Bak is associated with the microtubule pellet, whereas Bid remains primarily with the unpolymerized fraction. These interactions require the presence of the anionic C-termini of alpha- and beta-tubulin as they do not occur with tubulin S in which the C-termini have been removed. While in no way ruling out other pathways, such direct associations are the simplest potential regulatory mechanism for apoptosis resulting from disturbances in microtubule or tubulin function.     

Microtubule assembly affected by the presence of denatured tubulin

The effects of denatured tubulin on microtubule assembly from active phosphocellulose-tubulin have been studied. The presence of denatured tubulin resulted in an inhibition of the assembly and in the increase of the critical concentration to trigger the assembly. Inhibition of both the rate and extent of microtubule assembly was dependent on denatured tubulin concentration. This perturbation of microtubule assembly by denatured tubulin is likely to be specific as non-microtubule proteins did not significantly affect the assembly.     

Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

Interaction of rat brain microtubule proteins and 6S tubulin with rabbit skeletal muscle actomysin.

The interaction between actomyosin from rabbit skeletal muscle and microtubule proteins or 6S tubulin from rat brain was investigated with respect to the change in ATPase activity and physicochemical properties. Myosin bound to both microtubule proteins and 6S tubulin at low ionic strength. In the aggregates the molar ratio of microtubule proteins or 6S tubulin to myosin was 0.5–1.5 or 1.5–2.5. The superprecipitation of actomyosin was inhibited by 6S tubulin. The degree of superprecipitation inhibition was dependent on the mixing order of myosin, actin, 6S tubulin, and ATP. When myosin was preincubated first with 6S tubulin, the inhibition was most marked. The actin activation of myosin Mg-ATPase was inhibited by both microtubule proteins and 6S tubulin with stronger effects by the latter. The preincubation of myosin with 6S tubulin prior to the addition of actin induced not only greater inhibition of ATPase but also the binding of a larger quantity of 6S tubulin to myosin than the preincubation of myosin with actin. The similar results were obtained with microtubule proteins.     

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.     

The ins and outs of tubulin acetylation: more than just a post-translational modification?

Microtubules are highly dynamic polymers of α/β tubulin heterodimers that play key roles in cell division and in organizing cell cytoplasm. Although they have been discovered more than two decades ago, tubulin post-translational modifications recently gained a new interest as their role was increasingly highlighted in neuron differentiation and neurodegenerative disorders. Here, we specifically focus on tubulin acetylation from its discovery to recent studies that provide new insights into how it is regulated in health and disease and how it impacts microtubule functions. Even though new mechanisms involving tubulin acetylation are regularly being uncovered, the molecular links between its location inside the microtubule lumen and its regulators and effectors is still poorly understood. This review highlights the emerging roles of tubulin acetylation in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signalling. It also points out that tubulin acetylation should no longer be seen as a passive marker of microtubule stability, but as a broad regulator of microtubule functions.     

Impact of the ‘tubulin economy’ on the formation and function of the microtubule cytoskeleton

The microtubule cytoskeleton is assembled from a finite pool of α,β-tubulin, the size of which is controlled by an autoregulation mechanism. Cells also tightly regulate the architecture and dynamic behavior of microtubule arrays. Here, we discuss progress in our understanding of how tubulin autoregulation is achieved and highlight work showing that tubulin, in its unassembled state, is relevant for regulating the formation and organization of microtubules. Emerging evidence suggests that tubulin regulates microtubule-associated proteins and kinesin motors that are critical for microtubule nucleation, dynamics, and function. These relationships create feedback loops that connect the tubulin assembly cycle to the organization and dynamics of microtubule networks. We term this concept the ‘tubulin economy’, which emphasizes the idea that tubulin is a resource that can be deployed for the immediate purpose of creating polymers, or alternatively as a signaling molecule that has more far-reaching consequences for the organization of microtubule arrays.     

Estimation of the diffusion-limited rate of microtubule assembly.

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth.