一种改进的观察植物细胞微管的免疫荧光染色方法

植物微管体积小,而且始终处于动态变化之中,因此观察到清晰的微管形态有一定的难度。本文以拟南芥为实验材料,介绍了一种改进的植物细胞微管免疫荧光染色方法,用此方法可以观察到清晰、完整的植物微管形态,此法对其他植物细胞微管观察可能也有借鉴和参考价值。     

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。     

慈姑根尖细胞微管骨架的免疫荧光观察

分别用考马斯亮蓝染色和间接免疫荧光标记,并运用荧光倒置显微镜和激光共聚焦显微镜,对慈姑根尖固定后酶解获得的去壁细胞和细胞团块以及根尖细胞分裂周期中微管骨架列阵进行详细观察,以探索高等植物微管周期的普遍性。结果表明:慈姑根尖固定后酶解可获得大量结构完整的去壁细胞与细胞团块;考马斯亮蓝染色观察可见,慈姑根尖细胞中丰富的蛋白物质以及处于不同分裂期的细胞核染色体;免疫荧光观察可见,慈姑根尖细胞周期中微管骨架保存较好,主要有周质微管、早前期带微管、纺缍体微管和成膜体微管4种循序变化的排列方式,构成了高等水生植物分裂细胞中典型的微管周期。实验结果证明,高等水生植物与陆生植物微管周期具有相似性,为植物微管周期概念提供了新的实例。     

割手密茎尖细胞有丝分裂过程中微管骨架的变化

利用冰冻切片法结合间接免疫荧光标记技术对割手密茎尖细胞有丝分裂过程中微管骨架的变化进行了研究。结果表明:在割手密茎尖细胞有丝分裂过程中存在4种循序变化的典型微管列阵,即周质微管、早前期微管带、纺锤体微管及成膜体微管。在割手密初生增粗分生组织细胞中观察到的大多数是周质微管列阵,很少观察到其它3种典型的微管列阵,这可能这是割手密茎较小的原因之一。     

川百合花粉管的生殖细胞分裂过程中微管骨架的分布变化

应用透射电镜辅以免疫荧光定位技术研究了川百合 (Lilium davidii Duch.)花粉管中生殖细胞分裂过程中染色体动态和微管分布的关系。在生殖细胞分裂前和有丝分裂前期 ,电镜观察一直未见微管结构 ,但免疫荧光图象显示生殖细胞中有微管蛋白存在。直到分裂的前中期—中期 ,染色体出现 ,它们沿花粉管的长轴前后排列 ,横向的着丝点对相应地一对对地纵向排列。这时 ,生殖细胞中才出现大量微管 ,它们分布于细胞周质区和染色体之间 ,并跨越染色体的整个长度。前中期—中期开始时 ,只有 1～ 2对着丝点从横向转为纵向 ,微管垂直插入着丝点形成着丝点微管 ,而非前人用免疫荧光方法观察到的微管与着丝点侧向联接的图象。随着横向的着丝点对逐渐转变成纵向的过程 ,着丝点微管数量逐渐增多 ,但不形成典型的纺锤体。分裂后期 ,染色体交错分离 ,微管的分布与前中期—中期的基本相同。晚后期 ,染色体呈明显的两群 ,除极区和细胞中央区有微管残余外 ,大部分微管消失。通过染色体长度的测量 ,间接证明了分裂后期 B的存在。分裂末期的晚期 ,核膜形成后 ,在两精核之间的区域 ,微管数量开始增多。此区可能代表用免疫荧光所观察到的微管重叠区。细胞板出现后 ,微管消失     

神经元微管蛋白的研究进展

神经元特殊形态的形成及维持主要依赖于神经元细胞骨架中微管的装配,在此过程中,涉及到微管的组成及其动力学性质,而最终形成了稳定的微管结构,在神经元中,这一结构为沿着神经突运输物质提供了基础。本文将主要在神经元微管的结构与功能,神经元微管蛋白异构基因的表达及其翻译后加工形式等方面的研究进展加以综述。     

萱草花粉微管蛋白的体外聚合及电镜观察

微管(microtubule)作为细胞骨架的主要成分,在植物体内,微管除决定细胞的形状外,还参与很多重要的细胞功能。但有关微管蛋白生物化学的研究绝大多数来自动物脑组织材料,对植物微管蛋白的研究除培养细胞外所知甚少,我们纯化了毫克数量的萱草(Hemer-ocallis fulvaL.)花粉微管蛋白,利用紫杉醇作为促进剂,在Mg2 、GTP等存在下体外聚合成功,并观察了其电镜下的形态。     

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.     

微管与微管蛋白概述及其研究进展

本文综合了近年来有关微管、微管蛋白的研究进展,介绍了 MT 与微管蛋白的形态构造和生化特征;着重讨论了体内和离体条件下MT 的聚合过程,以及影响聚合的各种因素,如 MAP 和 Tau 蛋白等。最后简单地归纳了一下 MT 与其他细胞器的关系,以及 MT 的功能。MT 是如何由微管蛋白聚合成的,是目前MT 研究的关键。     

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。     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

Identifying the site of microtubule polymerization during regrowth of UV-sheared kinetochore fibres using antibodies against acetylated alpha-tubulin

Areas of reduced birefringence (ARBs) produced on chromosomal fibres of crane-fly spermatocyte spindles by ultraviolet microbeam irradiation move poleward. The ARB is due to the depolymerization of the microtubules in that area, and its poleward motion is due in part to the lengthening of that part of the kinetochore fibre which is left attached to the kinetochore after shearing the microtubules. We tested whether the lengthening of this fibre is due to the polymerization of microtubules at the growing edge of the ARB by staining growing fibres in irradiated spindles with antibodies to tubulin and to acetylated tubulin. We have previously argued that newly-polymerized kinetochore microtubules are not acetylated, whereas older kinetochore microtubules are (Wilson & Forer, 1989). Therefore we expected to see an absence of staining with antibodies to acetylated tubulin at the edge of the ARB if microtubules were polymerizing there. There is no absence of staining, however, which suggests that growth of the sheared microtubules does not occur at the ARB edge. Other possibilities are discussed.     

Electron microscope demonstration of tubulin in cilia and basal bodies of rat tracheal epithelium by the use of an antitubulin antibody

It has been previously demonstrated that both cytoplasmic microtubules and the microtubules of cilia, flagella, and sperm tail contain tubulin. Although the morphology of cytoplasmic microtubules and that of axonemes differs in cells from which they have been isolated, the tubulin of the two structures shares physical and chemical properties. In some mammalian tissues, such as tracheal epithelium, cilia and basal bodies are difficult to isolate and characterize. The use of an enzyme- labeled immunoglobulin probe would facilitate identification and in situ localization of such proteins. Tubulin prepared from porcine brain by ion-exchange chromatography and from rat brain by the method of cyclic polymerization and depolymerization with subsequent disk gel electrophoresis with SDS were injected intravenously into rabbits. The animals were intermittently bled and the antisera extracted. The specificity of the antisera was proved by indirect immunofluorescence staining of the mitotic spindle, specific blocking of spindle staining by purified tubulin and not by other proteins, staining of 3T3 cytoplasmic microtubules, single line on immunoelectrophoresis, failure of control antisera to show any of these, and precipitation of antibody with all tubulin preparations and not with actin. We have shown by electron microscopy of ciliated cells of the tracheal epithelium stained with antitubulin by the indirect enzyme-labeled antibody method that the basal bodies, outer doublets, and central pair of the cilia contain tubulin. This indicates that tubulin in microtubules of cilia and basal bodies of rat tracheal epithelium is antigenically similar to tubulin extracted from cytoplasmic neurotubules of brains from the same species and from a different mammalian species. No other axonemal structures stained with the antitubulin. Three different preparations of tubulin from pigs and rats were used to immunize rabbits. All elicited similar antisera which gave identical staining patterns. The specificity of the staining was demonstrated by the absence of staining with immune serum absorbed with purified tubulin, the absence of staining with preimmune serum, and the absence of staining if any of the reagents were omitted during the staining reaction.     

Microtubules in the neurosecretory neurones of the posterior pituitary of the rat

Summary The microtubules in the neurosecretory neurones of the posterior pituitary were studied using different electron microscopical techniques. Tannic acid staining indicated that the microtubules had a 13 protofilament substructure similar to that described for microtubules from other tissues and organisms; the dimensions of the microtubules were also similar to that previously reported. Albumen pretreatment clearly showed the microtubules running across axonal swellings, but not continuing across the nerve endings. The only organelles showing possible association with the microtubules were small vesicles and smooth endoplasmic reticulum, no association between hormone granules and microtubules could be seen. Acknowledgement: The author is grateful to P. Wilks for skilled technical assistance.     

Early events in rice somatic embryogenesis: Changes in cortical microtubule distribution and orientation

Summary The changes in the microtubular pattern during the first few days of somatic embryo induction were studied using the rice scutellum epithelium system. A clear difference in the distribution and orientation of the microtubules could be found as early as 12 h after culturing. In the control treatment, the microtubules tended to congregate near the terminal part of the cells and had an oblique to transverse orientation with respect to the long axis of the cells. In the hormone-treated explants, the microtubules were more evenly distributed throughout the cells as compared to the control treatment. The microtubules initially had an oblique orientation similar to the control. However, by 16 h, the majority of microtubules were aligned parallel to the long axis of the cells. The scutellum epithelial cells in both the control- and hormone-treated explants divided periclinally at about 48 h. In the control treatment, the scutellum epithelial cells gradually became vacuolated over time, while the hormone-treated explants remained densely cytoplasmic. The fluorescence staining intensity gradually declined in the control, whereas a distinct staining pattern of microtubules remained in the hormone-treated explants.     

Distribution of microtubules and microfilaments in thyroid follicular epithelial cells of normal,TSH-treated,aged, and hypophysectomized rats

Summary We investigated the distribution of microtubules and microfilaments in rat thyroid follicular epithelial cells by applying an immunofluorescence technique with monoclonal antibodies against tubulin and by staining sections with rhodamine-phalloidin. In normal thyroid cells, microtubules run longitudinally from the apical region to the basal region intersecting with each other. In addition, intense labelling with tubulin antibodies was observed in the apical part of the cell. The ultrastructural examinations showed that microtubules often run along the apical plasma membrane. Dot-like labelling with anti-tubulin antibodies was often observed in the perinuclear space, but no microtubules were recognized in the nucleus. Microfilaments bound to rhodamine-phalloidin were distributed mainly beneath the apical plasma membrane, and the portion along the basolateral membrane was scarcely positive. The apical pole of the follicle cell was also decorated by anti-microtubule-associated protein-2 (MAP-2). After TSH stimulation, the intensity of immunocytochemical staining against tubulin was remarkably increased in the cytoplasm. Simultaneously, at the apical region, the staining intensity of rhodamine-phalloidin was increased. Microtubules and microfilaments appeared in the pseudopods after TSH injection. In hypophysectomized or aged rats, thyroid follicular epithelial cells decreased in height, and both immunofluorescent labelling against tubulin and rhodamine-phalloidin labelling were markedly decreased. These results indicate that the distribution and polymerization of microtubules and microfilaments in thyroid follicular epithelial cells vary with the functional stage.     

Endoplasmic Reticulum to Golgi Trafficking in Multinucleated Skeletal Muscle Fibers

The organization of membrane trafficking between endoplasmic reticulum and Golgi within multinucleated muscle fibers was analyzed. We found that markers for the compartment involved in endoplasmic reticulum to Golgi trafficking exhibited perinuclear as well as interfibrillar localization. Furthermore, these markers showed prominent colocalization with microtubules. To analyze membrane trafficking, we followed the temperature-controlled transport of the G protein of the mutant vesicular stomatitis virus, tsO45, in isolated myofibers. Perinuclear and cross-striated staining were seen at 39°C, while at 15°C a diffuse staining component appeared along a subset of interfibrillar microtubules. At 20°C, bright Golgi spots were seen to be associated with microtubules that appeared as circumnuclear rings and longitudinal bundles. Beneath the motor end plate, however, the organization of the Golgi elements and microtubules was found to be distinctive. Retrograde trafficking induced by brefeldin A resulted in the disappearance of the Golgi spots throughout the myofibers and the appearance of staining along microtubules. Thus, interfibrillar membranes seem to be active in protein export, and trafficking between endoplasmic reticulum and Golgi elements occurred throughout the myofibers. The results suggest that microtubules served as tracks for the two-way trafficking between the endoplasmic reticulum and the Golgi compartment.     

Microtubule distribution during fertilization in the rabbit.

The distribution of microtubules was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti-alpha-tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronuclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.     

Synapsin-1 is found in a microtubule-associated complex of proteins isolated from bovine brain

We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.     

Differences in surface morphology of microtubules reconstituted from pure brain tubulin using two different microtubule-associated proteins: the high molecular weight MAP 2 proteins and tau proteins.

Microtubules were reconstituted from homogeneous brain tubulin and homogeneous preparations of two different microtubule associated proteins, the high molecular weight MAP 2 proteins or the tau proteins. The resulting microtubules were characterized by three electron microscopical procedures: Thin sectional analysis of embeded material, negative staining analysis using a STEM microscope and high resolution metal-shadowing analysis. By all three procedures MAP 2 microtubules have a much rougher surface morphology than tau microtubules, in agreement with the much higher molecular weight of the MAP 2 proteins. Tau microtubules, however, do not show the very smooth surface of microtubules assembled from pure tubulin in the absence of any microtubule associated proteins. In the case of MAP 2 microtubules thin sectional analysis as well as metal shadowing reveals that the globular protrusions seen in negative staining analysis appear as linear side arms which may extend by as much as 30 nm on both sides from the microtubular wall proper, giving rise to an overall structure with a diameter close to 100 nm. The possible implication of such structures for in vivo situations is briefly discussed as is the possibility that the "halo-effect" around microtubules seen in vivo may be due to a structural organization similar to that of MAP 2 tubules in vitro.