Positioning of the mitotic spindle by a cortical-microtubule capture mechanism

Correct positioning of the mitotic spindle is critical for cell division and development. Spindle positioning involves a search-and-capture mechanism whereby dynamic microtubules find and then interact with specific sites on the submembrane cortex. Genetic, biochemical, and imaging experiments suggest a mechanism for cortical-microtubule capture. Bim1p, located at microtubule distal ends, bound Kar9p, a protein associated with the daughter cell cortex. Bim1p is the yeast ortholog of human EB1, a binding partner for the adenomatous polyposis coli tumor suppressor. EB1 family proteins may have a general role in linking the microtubule cytoskeleton to cortical polarity determinants.     

甘蔗茎尖细胞有丝分裂过程中微管骨架的变化（英文）

In order to understand the microtubule change of monocotyls stem-tip during mitosis,the arrangement,transformation of microtubule array and its relation with chromosome movement during mitosis were studied with freezing microtome,indirect immunofluorescence,DAPI staining and fluorescence microscopy.The results showed that nucleolus was intact when the cortical microtubules formed;cortical microtubules were changed into phramoplast microtubule bands at mitosis prophase.When phramoplast microtubule came into being,nuclear membrane was ruptured and chromosome was arranged at the position of cell plate;subsequently,phramoplast microtubules were changed into phragmoplast microtubules,phramoplast microtubules were shortening and microtubules on the sides of cell plate were increasing gradually,during this course sister chromatid was separated by microtubules at cell plate and tract to the two poles,forming phragmoplast microtubules.Then the nucleolus of two daughter cells formed and separated in the end with the increase of cells numbers.Therefore,cell division orientation could be judged from the arrangement of cell microtubules in different periods in order to understand its growth status.     

甘蔗茎尖细胞有丝分裂过程中微管骨架的变化

[目的]研究单子叶植物茎尖有丝分裂细胞的微管动态变化情况。[方法]采用冰冻切片法结合间接免疫荧光技术及DAPI染色,利用荧光显微镜观察甘蔗茎尖细胞有丝分裂时微管列阵的排列、转换及与染色体运动的关系。[结果]当周质微管形成时,细胞核保持完整;有丝分裂前期,周质微管转变为早前期微管带;当纺锤体微管形成时,细胞核膜破裂,染色体排列在细胞板位置;之后纺锤体微管向成膜体微管转换,纺锤体微管逐渐缩短而细胞板两侧微管逐渐增加,在这个过程中姊妹染色体被微管从细胞板处分离并牵引至两极,从而形成成膜体微管;之后两个子细胞的细胞核形成,并最终分裂,细胞数量增加。[结论]从细胞微管各时期的排列就可以判断出细胞分裂方向,了解其生长情况,为甘蔗增粗机理的研究提供例证。     

甘蔗茎尖细胞有丝分裂过程中微管骨架的变化

笔者拟从微管列阵变化及其参与染色体运动的关系方面探讨甘蔗茎增粗机理,为单子叶植物的微管与染色体相关研究提供一定的例证。     

Dynamic instability of sheared microtubules observed by quasi-elastic light scattering

The kinetics of microtubule reassembly was studied in vitro by quasi-elastic light scattering (QELS). When microtubules assembled in the absence of microtubule-associated proteins (MAPs) were sheared, they rapidly depolymerized, recovered, and reassembled. The mean length of the recovered microtubules was the same as that observed just before shearing, implying that on average one fragment per original microtubule survived the fragmentation and recovery. When microtubules that contained 25 percent brain MAP were sheared, the fragments did not depolymerize extensively and the average length of the fragments decreased by a factor of 3 relative to the unsheared sample. The results support the dynamic instability model, which predicts that cellular microtubules are latently unstable structures protected on their ends by stabilizing caps.     

Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues

Microtubules display dynamic instability, with alternating phases of growth and shrinkage separated by catastrophe and rescue events. The guanosine triphosphate (GTP) cap at the growing end of microtubules, whose presence is essential to prevent microtubule catastrophes in vitro, has been difficult to observe in vivo. We selected a recombinant antibody that specifically recognizes GTP-bound tubulin in microtubules and found that GTP-tubulin was indeed present at the plus end of growing microtubules. Unexpectedly, GTP-tubulin remnants were also present in older parts of microtubules, which suggests that GTP hydrolysis is sometimes incomplete during polymerization. Observations in living cells suggested that these GTP remnants may be responsible for the rescue events in which microtubules recover from catastrophe.     

The kinesin Klp2 mediates polarization of interphase microtubules in fission yeast

Fission yeast (Schizosaccharomyces pombe) cells grow longitudinally in a manner dependent on a polarized distribution of their interphase microtubules. We found that this distribution required sliding of microtubules toward the cell center along preexisting microtubules. This sliding was mediated by the minus end-directed kinesin motor Klp2, which helped microtubules to become properly organized with plus ends predominantly oriented toward the cell ends and minus ends toward the cell center. Thus, interphase microtubules in the fission yeast require motor activities for their proper organization.     

Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardi

Basal bodies isolated from Chlamydomonas reinhardi will serve as initiation centers for the assembly of chick brain microtubule protein subunits (tubulin) into microtubules. The rate of microtubule assembly is tubulin-concentration dependent; this assembly occurs onto both distal and proximal ends of the basal body mnicrotubules, with distal assembly greatly favored. In vitro assembly of brain tubulin also occurs onto the mid-lateral aspects of the basal bodies, presumably onto the fiber connecting the two basal bodies.     

Differential regulation of dynein and kinesin motor proteins by tau

Dynein and kinesin motor proteins transport cellular cargoes toward opposite ends of microtubule tracks. In neurons, microtubules are abundantly decorated with microtubule-associated proteins (MAPs) such as tau. Motor proteins thus encounter MAPs frequently along their path. To determine the effects of tau on dynein and kinesin motility, we conducted single-molecule studies of motor proteins moving along tau-decorated microtubules. Dynein tended to reverse direction, whereas kinesin tended to detach at patches of bound tau. Kinesin was inhibited at about a tenth of the tau concentration that inhibited dynein, and the microtubule-binding domain of tau was sufficient to inhibit motor activity. The differential modulation of dynein and kinesin motility suggests that MAPs can spatially regulate the balance of microtubule-dependent axonal transport.     

Molecular linkage underlying microtubule orientation toward cortical sites in yeast

Selective microtubule orientation toward spatially defined cortical sites is critical to polarized cellular processes as diverse as axon outgrowth and T cell cytotoxicity. In yeast, oriented cytoplasmic microtubules align the mitotic spindle between mother and bud. The cortical marker protein Kar9 localizes to the bud tip and is required for the orientation of microtubules toward this region. Here, we show that Kar9 directs microtubule orientation by acting through Bim1, a conserved microtubule-binding protein. Bim1 homolog EB1 was originally identified through its interaction with adenomatous polyposis coli (APC) tumor suppressor, raising the possibility that an APC-EB1 linkage orients microtubules in higher cells.     

Inhibition of Plant Microtubule Polymerization in vitro by the Phosphoric Amide Herbicide Amiprophos-Methyl

The phosphoric amide herbicide amiprophos-methyl (APM) produced a concentration-dependent inhibition of taxol-induced rose microtubule polymerization in vitro. Parallel studies on taxol-induced assembly of bovine brain microtubules showed no effect of APM at a concentration ten times that required to give complete inhibition of rose microtubule assembly. The data indicate that (i) APM is a specific and potent antimicrotubule drug and (ii) APM directly poisons microtubule dynamics in plant cells, rather than indirectly depolymerizing microtubules through a previously proposed mechanism involving deregulation of intracellular calcium levels.     

Directional switching of the kinesin Cin8 through motor coupling

Kinesin motor proteins are thought to move exclusively in either one or the other direction along microtubules. Proteins of the kinesin-5 family are tetrameric microtubule cross-linking motors important for cell division and differentiation in various organisms. Kinesin-5 motors are considered to be plus-end-directed. However, here we found that purified kinesin-5 Cin8 from budding yeast could behave as a bidirectional kinesin. On individual microtubules, single Cin8 motors were minus-end-directed motors, whereas they switched to plus-end-directed motility when working in a team of motors sliding antiparallel microtubules apart. This kinesin can thus change directionality of movement depending on whether it acts alone or in an ensemble.     

Distinguishing inchworm and hand-over-hand processive kinesin movement by neck rotation measurements

The motor enzyme kinesin makes hundreds of unidirectional 8-nanometer steps without detaching from or freely sliding along the microtubule on which it moves. We investigated the kinesin stepping mechanism by immobilizing a Drosophila kinesin derivative through the carboxyl-terminal end of the neck coiled-coil domain and measuring orientations of microtubules moved by single enzyme molecules at submicromolar adenosine triphosphate concentrations. The kinesin-mediated microtubule-surface linkage was sufficiently torsionally stiff (>/=2.0 +/- 0.9 x 10(-20) Newton meters per radian2) that stepping by the hypothesized symmetric hand-over-hand mechanism would produce 180 degree rotations of the microtubule relative to the immobilized kinesin neck. In fact, there were no rotations, a finding that is inconsistent with symmetric hand-over-hand movement. An alternative "inchworm" mechanism is consistent with our experimental results.     

A TOG:αβ-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase

Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use αβ-tubulin-interacting tumor overexpressed gene (TOG) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin, but the mechanism by which they do so has remained unclear. Here, we report the structure of the TOG1 domain from Stu2p bound to yeast αβ-tubulin. TOG1 binds αβ-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a curved conformation of αβ-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with αβ-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of αβ-tubulin and how they selectively recognize the growing end of the microtubule.     

Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host Pathogen Sphaerotheca fuliginea

The plant cytoskeleton is a highly dynamic and versatile intracellular scaffold composed of microtubules and microfilaments, serving a multiplicity of functions in plant cells. To reveal the relationship between the cytoskeleton in wheat （Triticum aestivum L.） cv. Suwon 11 attacked by the non-host pathogen Sphaerotheca fuliginea and the initiation of the hypersensitive response, the microtubule inhibitor oryzalin was injected into the wheat leaves immediately prior to inoculation. The incidence of hypersensitive cell death was significantly lower than that in water-treated control. In addition, the occurrence of hypersensitive cell death was also delayed and S. fuliginea was able to penetrate and form haustoria in epidermal tissues of wheat. All the results above indicated that hypersensitive cell death was associated with depolymerisation of microtubules, suggesting that microtubules might play an important role in the expression of non-host resistance of wheat.     

Spatial patterns from oscillating microtubules

Microtubules are fibers of the cytoskeleton involved in the generation of cell shape and motility. They can be highly dynamic and are capable of temporal oscillations in their state of assembly. Solutions of tubulin (the subunit protein of microtubules) and guanosine triphosphate (GTP, the cofactor required for microtubule assembly and oscillations) can generate various dissipative structures. They include traveling waves of microtubule assembly and disassembly as well as polygonal networks. The results imply that cytoskeletal proteins can form dynamic spatial structures by themselves, even in the absence of cellular organizing centers. Thus the microtubule system could serve as a simple model for studying pattern formation by biomolecules in vitro.     

Preferred microtubules for vesicle transport in lobster axons

The hypothesis that transported vesicles are preferentially associated with a subclass of microtubules has been tested in lobster axons. A cold block was used to collect moving vesicles in these axons; this treatment caused the vesicles to accumulate in files along some of the microtubules. Quantitative analysis of the number of vesicles associated with microtubule segments indicated that lobster axons have two distinct populations of microtubules--transport microtubules that are the preferred substrates for vesicle transport and architectural microtubules that contribute to axonal structure.     

Microtubule-macrotubule transitions: intermediates after exposure to the mitotic inhibitor vinblastine

Vinblastine treatment of microtubule protein or intact microtubules assembled in vitro produced bifilar rings and bifilar helices. Suspentsions of rings and helices were demonstrated to bind [(3)H]colchicine, a diagnostic property of microtubule protein. Macrotubules are suggested to consist of tightly coiled helices formed by longitudinal compacting of loosely coiled protofilament pair intermediates.     

稻水象甲精子尾部的超微结构

为了解稻水象甲(Lissorhoptrus oryzophilus Kuschel)精子形成过程,通过扫描电镜照片观察其精子形成过程中部分时期的尾部超微结构.发现该虫精子鞭毛内部由2个线粒体衍生物、2个副体、1个中心粒侧体和1个微管系统(轴丝)构成,轴丝由9个副微管、9对双微管和2个中央微管组成.属典型的9+9+2构型.在精子形成过程中,两线粒体衍生物的形态和内容物有显著差异.中心粒侧体在后期才出现,包围线粒体衍生物、副体、中心粒侧体的微管在后期则全部消失.表明稻水象甲精子的形成过程相似于已报道的一些鞘翅目昆虫,但在线粒体衍生物、中心粒侧体、微管的发生上有其一定的特殊性.     

Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor

Completion of cell division during cytokinesis requires temporally and spatially regulated communication from the microtubule cytoskeleton to the actin cytoskeleton and the cell membrane. We identified a specific inhibitor of nonmuscle myosin II, blebbistatin, that inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly. Using blebbistatin and other drugs, we showed that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis. Continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.