甘蔗茎尖细胞有丝分裂过程中微管骨架的变化

利用改进的冰冻切片法结合间接免疫荧光标记技术对甘蔗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,在甘蔗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。同时,还观察到在各种典型微管列阵相互转变过程中存在各种微管列阵的过渡状态。甘蔗茎尖正在伸长的幼叶部位细胞的周质微管主要为与细胞伸长轴相垂直的横向周质微管：茎尖幼叶部位伸长缓慢细胞的微管主要为纵向及斜向排列的周质微管,在甘蔗茎尖幼叶基部初生增粗分生组织处,横向、斜向、纵向及随机排列的周质微管列阵均有分布。在少数分裂前期的细胞中,发现细胞具有2条早前期微管带,其具体功能还不清楚。表明甘蔗茎尖细胞微管列阵的变化与许多双子叶植物及部分单子叶植物具有共同的变化规律,进一步证明微管骨架的周期性变化在植物中具有普遍性。     

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。     

冰冻切片法在植物微管骨架研究中的应用

介绍了冰冻切片法研究植物微管骨架的一般程序和技术上的一些改进,结果证明,改进的冰冻切片技术,可以对植物不同类型的细胞进行很好的标记。实验结果表明,甘蔗正在迅速伸长的幼叶分布的微管类型主要是与细胞伸长轴方向垂直的周质微管,幼叶基部尤其是第三幼叶基部分布的主要是与细胞伸长轴方向平行的周质微管。表明冰冻切片法在植物微管骨架的研究中具有广阔的应用前景。     

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     

大蒜根尖细胞微管的免疫荧光观察

1963年,先后在动物和高等植物细胞中发现微管结构。已经知道微管不仅具有支持功能,而且在运动、运输和分泌等一系列细胞活动中发挥重要作用。在高等植物细胞中,微管明显地参与形态建成。周质微管(Cortical microtubules)可能与细胞壁中纤维素微纤丝的排列与定向有关。早前期带(Preprophase Bands)预示胞质分裂时细胞板的位置。成膜体微管参     

小麦叶片细胞周质微管的研究

采用铜网粘附-负染色法,并结合超薄切片,对小麦幼叶和成熟叶片细胞内的周质微管进行了研究,结果如下: (1) 粘附于铜网支持膜上的质膜片段,往往包含一个组织中心的微管体系。微管组织中心具有电子致密度很高的浓密物质。微管从组织中心呈辐射状或扇形分布。微管之间,有单个或数根成束排列, 有的相互平行,有的则相互交叉形成网状结构。微管的外径为24—24.76毫微米,最大长度为12微米。(2) 周质微管与质膜之间有密切联系,两者之间有连丝结构(“桥”)相连接。微管-桥-质膜三者结合形成一个稳定的体系。(3) 不仅质膜能粘附于铜网的福尔马支持膜上,分离原生质体残留的细胞壁纤维素微丝也能粘附于其上。被粘附的网状排列的纤维素微丝与幼叶细胞中周质微管的网状排列相一致,说明周质微管与纤维素微丝排列方向的密切关系。(4) 正在迅速生长的幼叶细胞比成熟叶片具有更多的周质微管和小泡结构(Vesicles),显示这两种细胞器的数量与细胞生长及细胞壁增生加厚的活动强度成正相关。     

粗茎鳞毛蕨原叶体细胞有丝分裂过程中微管列阵的变化

应用Steedman‘s wax切片法,间接免疫荧光标记技术和激光共聚焦扫描显微镜技术研究了粗茎鳞毛蕨（Dryopteris crassirhizoma Nakai)原叶体大液泡化细胞和分生组织细胞有丝分裂过程中微管列阵的变化。结果显示：应用高浓度的多聚甲醛（8%）可以很好地保持大液泡化细胞的结构和微管的抗原性。结果也显示Steedman‘s wax切片法和间接免疫荧光标记技术的优点;（1）避免在微管标记过程中酶解细胞壁;（2）在乙醇脱水过程中样品中叶绿素的自发荧光被减到最小;（3）能够详细观察到有丝分裂过程中微管骨架的变化。因此,这种方法可以被广泛用来调查简单植物体和复杂植物体中细胞的有丝分裂过程以及发育过程中微管骨架的变化。     

粗茎鳞毛蕨原叶体细胞有丝分裂过程中微管列阵的变化

粗茎鳞毛蕨原叶体细胞有丝分裂过程中微管列阵的变化 何群 尤瑞麟 姆旺戈     

百合生殖细胞分裂过程中微管分布的显微荧光观察

用抗微管蛋白抗体和荧光标记技术,观察了百合生殖细胞经有丝分裂形成精细胞过程中微管的变化。生殖细胞在分裂的前期,存在于核外围以及细胞两端胞质内的微管大都以微管束的形式沿细胞长轴方向平行排列。在靠近核的部位,有些微管有时会斜向排列。分裂进入中期后,染色体集中排列在赤道面。在染色体周围可以见到有多束与细胞长轴平行排列着的微管,但这些微管束是在分裂中期时新形成的或是在前期已存在,尚难以断定。这些微管束有一个特点,就是当它们延伸至赤道板部位时,在每一条微管束上都有一个无荧光的小圆区;这个小圆区可能代表着丝粒的位置。细胞分裂进入后期,姊妹染色单体分别向两极移动形成两组染色体。在它们之间近赤道板位置出现了一个具有强烈荧光的区域,显示在这一部位,微管相当浓密。从这一强烈荧光区向两极分别伸出多条微管束。因此,在这一强烈荧光区内可能有多个微管束重叠。到细胞分裂末期,在这一强烈的荧光区的中央出现了一条横向的无荧光区。这一区域有可能为胞质完成分裂后新形成的细胞板所在的部位。     

微管骨架在轮藻节间细胞伸长生长中的作用

利用免疫荧光定位及激光共聚焦扫描显微镜,结合细胞生长曲线的定量测定,对不同生长阶段的轮藻节间细胞微管骨架进行了观察研究,结果如下:轮藻顶端生长活跃的新生细胞中,与细胞长轴垂直的周质微管(cortical microtubules)占绝对优势,随着生长速率的减慢,周质微管由垂直于细胞长轴逐渐转为平行排列;基部生长基本停止的节间细胞中,胞内微管则以平行细胞长轴为主;不同生长阶段节间细胞的微管骨架,对微管特异解聚剂黄草消(oryzalin)处理的敏感性表现不相同。顶端生长活跃的节间细胞经oryzalin处理40min后,绝大多数周质微管发生解聚;而基部生长基本停止的老细胞中,即使延长处理时间,仍残留一些尚未完全解聚的微管片段;10μmol/L微管解聚剂oryzalin处理轮藻顶端新生细胞,在高精度的细胞伸长生长测定装置监测下,发现oryzalin对细胞的伸长生长速率有明显的抑制作用,去掉药剂后,伸长生长又有一定的恢复。并且发现,经o-ryzalin处理后,微管的解聚(40min左右)与顶端节间细胞伸长生长的停止(100min左右)两者间存在着时间上的差异,即微管解聚在先,细胞伸长停止在后。以上结果均说明微管骨架在轮藻节间细胞生长中具有重要作用。     

Orientation of cortical microtubules correlates with cell shape and division direction

Summary Cortical microtubules in the epidermis of regeneratingGraptopetalum plants were examined by in situ immunofluorescence. Paradermal slices of tissue were prepared by a method that preserves microtubule arrays and also maintains cell junctions. To test the hypothesis that cortical microtubule arrays align perpendicular to the direction of organ growth, arrays were visualized and their orientation quantified. A majority of microtubules are in transverse orientation with respect to the organ axis early in shoot development when the growth habit is uniform. Later in development, when growth habit is non-uniform and the tissue is contoured, cortical microtubules are increasingly longitudinal and oblique in orientation. Microtubules show only a minor change in orientation at the site of greatest curvature, the transition zone of a developing leaf. To assess the role of the division plane on orientation of arrays, the pattern of microtubules was examined in individual cells of common shape. Cells derived from transverse divisions have predominately transverse cortical arrays, whereas cells derived from oblique and longitudinal divisions have non-transverse arrays. The results show that, regardless of the stage of development, microtubules orient with respect to cell shape and plane of division. The results suggest that cytoskeletal function is best considered in small domains of growth within an organ.Abbrevations DMSO dimethylsulfoxide - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetra acetic acid - FITC fluorescein isothiocyanate - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Microtubule organization in three-dimensional confined geometries: evaluating the role of elasticity through a combined in vitro and modeling approach

Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a confining box-like geometry, in the absence of other (active) microtubule organizing processes. This is inspired, for example, by the cortical microtubule array of elongating plant cells, where microtubules are typically organized in an aligned array transverse to the cell elongation axis. The method we adopt is a combination of analytical calculations, in which the polymers are modeled as inextensible filaments with bending elasticity confined to a two-dimensional surface that defines the limits of a three-dimensional space, and in vitro experiments, in which microtubules are polymerized from nucleation seeds in microfabricated chambers. We show that these features are sufficient to organize the polymers in aligned, coiling configurations as for example observed in plant cells. Though elasticity can account for the regularity of these arrays, it cannot account for a transverse orientation of microtubules to the cell's long axis. We therefore conclude that an additional active, force-generating process is necessary to create a coiling configuration perpendicular to the long axis of the cell.     

Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules

Mitochondrion movement and positioning was studied in elongating cultured cells of tobacco (Nicotiana tabacum L.), containing mitochondria-localized green fluorescent protein. In these cells mitochondria are either actively moving in strands of cytoplasm transversing or bordering the vacuole, or immobile positioned in the cortical layer of cytoplasm. Depletion of the cell's ATP stock with the uncoupling agent DNP shows that the movement is much more energy demanding than the positioning. The active movement is F-actin based. It is inhibited by the actin filament disrupting drug latrunculin B, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime and the sulphydryl-modifying agent N-ethylmaleimide. The microtubule disrupting drug oryzalin did not affect the movement of mitochondria itself, but it slightly stimulated the recruitment of cytoplasmic strands, along which mitochondria travel. The immobile mitochondria are often positioned along parallel lines, transverse or oblique to the cell axis, in the cortical cytoplasm of elongated cells. This positioning is mainly microtubule based. After complete disruption of the F-actin, the mitochondria parked themselves into conspicuous parallel arrays transverse or oblique to the cell axis or clustered around chloroplasts and around patches and strands of endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel arrays.     

Microtubule orientation and dynamics in elongating characean internodal cells following cytosolic acidification, induction of pH bands, or premature growth arrest

Summary Cortical microtubules (MTs) at indifferent zones in immatureNitella internodes were investigated by injection of fluorescently tagged sheep brain tubulin into living cells and by immunofluorescence on fixed material. Nearly identical MT patterns and numbers were detected with the two techniques, indicating that sheep brain tubulin incorporated into all cortical MTs. MTs were aligned transversely to the long axis of the cell and approximately one MT was present every micrometer of longitudinal cell distance. Treatment of internodes with propionic acid to acidify cytosolic pH caused depolymerization of MTs and an increase in the unpolymerized tubulin pool. Transfer of young, vigorously elongating cells to media inducing premature growth cessation resulted in a slight decrease in microtubule numbers but did not significantly alter microtubule orientation patterns or microtubule lifespans. MTs remained transverse for days following growth cessation before finally assuming a more random alignment characteristic of mature, non-growing internodes. No differences in MT numbers, orientation, or dynamics were detected between acid and alkaline bands in internodes incubated in a band-inducing medium. Thus, properties of cortical MT arrays were not closely coupled to growth status or to regional differences in cellular physiology associated with pH banding.Abbrevations BIM band-inducing medium - CCM Chara culture medium - CF carboxyfluorescein - FRAP fluorescence redistribution after photobleaching - MT microtubule     

Microtubule rearrangements accompanying dedifferentiation in mesophyll cultures ofZinnia elegans L.

Summary To determine the orientation of cortical microtubule arrays in mesophyll cells ofZinnia, a new technique designed to increase the rate of fixation of excised leaf tissue and subsequent permeabilization of mesophyll cell walls was developed. This procedure resulted in immunolabeling of high percentages of mesophyll cells, making it possible to quantify cells with different types of cortical microtubule arrays. When developing palisade mesophyll cells were fixed in situ, most of the cells had cortical microtubules organized in parallel arrays oriented transverse to the long axis. Delay in the transfer of leaf tissue to fixative resulted in increased numbers of cells with random cortical microtubule orientations, indicating that arrays may become reoriented rapidly during leaf excision and cell isolation procedures. The role of wound-induced microtubule reorientation in mesophyll dedifferentiation and tracheary element development is discussed.Abbreviations BSA bovine serum albumin - CMT cortical microtubule - TE tracheary element - TBS tris-buffered saline     

Pressure Induced Reorientation of Cortical Microtubules in Epidermal Cells of Lolium rigidum Leaves

In order to assess the effect on microtubule arrays of slowlypressurising cells over 50 s from 0.1 MPa (atmospheric pressure)to 55 MPa, microtubules in epidermal cells of Lolium rigidumleaves were visualised by immunofluorescent staining and fluorescencemicroscopy. In both control and pressure-treated leaves cellshape, measured as the ratio of cell length and width, can becorrelated to the arrangement of cortical microtubules. Microtubulearrays change from random to organised in cells whose lengthis greater than their width. In untreated leaves, elongatedcells have microtubules aligned predominantly transversely.In pressure-treated leaves, elongated cells have microtubulesaligned predominantly longitudinally. Thus, pressure treatmentresults in the rapid reorientation of organised cortical microtubulesfrom a transverse to a longitudinal orientation. (Received June 21, 1993; Accepted July 15, 1993)