Microtubule dynamics.

Although compelling evidence has been obtained for heterogeneity in the structure of subunits in microtubules, it has not been possible to prove that this results from the presence of tubulin-GDP and tubulin-GTP in polymers. There are reasons to exclude the existence of even a monolayer of tubulin-GTP subunits at microtubule ends. Dynamic behavior appears to be best accounted for by a mechanism in which tubulin-GDP in microtubules exists in two conformations. The mechanism of microtubule-associated protein binding to microtubules and the role of phosphorylation on this reaction are discussed.     

Microtubule organization in lymphoid malignancies.

The abnormal organization of actin-containing microfilaments and vimentin-containing intermediate filaments in neoplastic lymphocytes of T and B cell origin has been described. We investigated microtubules of pathologic cells from 34 lymphoid malignancies, by immunofluorescence microscopy, using monoclonal tubulin antibody. In most cases, apart from two cases of lymphoma, one T cell lymphoma and one B cell lymphoma, interphase leukemia cells, lymphoma cells, and myeloma cells were shown to contain well-organized microtubules which were associated with a microtubule organization center at one end. In the cells of a patient with T cell lymphoma, although microtubules were not visible in the lymphoma cells from lymph nodes, they became visible after 72 hours in culture with concanavalin A (Con A) and interferon alpha. Cap formation was observed with antitubulin monoclonal antibody in the peripheral blood lymphocytes from a chronic lymphocytic leukemia patient, but well-developed microtubules were observed on other occasions in the same patient. There were no obvious structural differences between microtubules in T and B cell lymphoid malignancies, but leukemia cells and lymphoma cells with irregularly shaped nuclei, such as adult T cell leukemia cells and B cell lymphoma cells with cleaved nuclei, had complicated microtubules surrounding their irregular nuclei. In general, after blastogenic stimuli with phytohemagglutinin-P (PHA-P), Con A, and pokeweed mitogen (PWM), the development of the microtubules was proportional to the incorporation of 3H thymidine (3H-TDR). In most cases, after incubation with granulocyte colony-stimulating factor (G-CSF) and interferon alpha, the number of intact cells decreased and the number of degenerated cells increased, but the intact cells had intact microtubules.     

Microtubule dynamics

A combination of biochemical, structural, and morphological analyses during the last 2 decades has shown that the cytoplasm of a cell is not a disorganized mass of jelly but a highly structured cell compartment formed of a cytoskeleton, one of which principal components are the microtubules. More recently, studies have revealed that microtubule cytoskeleton is not only well organized but highly dynamic, and that microtubule dynamics may be responsible for several cell functions such as chromosome segregation, cell morphogenesis, or intracytoplasmic organization.     

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。     

Microtubule assembly in developing mammalian brain.

Microtubule protein was measured in mouse brain homogenates by quantitative colchicine binding. Neonatal animals contained more than twice the amount of brain tubulin as adult mice. The percentage of colchicine-binding protein which was polymerized was determined by extracting brain at room temperature into a medium designed to stabilize intact microtubules. Under identical conditions and tubulin concentrations, neonatal brain tubulin (colchicine-binding activity) had a greater proportion of the total extracted in an apparently polymerized state (pelletable by centrifugation) than did adult brain. A slight variation in the ratio of assembled to unassembled tubulin was observed with varying protein concentration (volume of extract), indicating that the values obtained may not reflect exactly the in vivo situation, because a rapid equilibration takes place upon homogenization. At all protein concentrations, the neonatal brain extracts contained a significantly greater proportion of assembled tubulin than did adult brain. This proportion began to fall at 5 days postnatal and reached the adult level at 30 days. The tubulin assembled/not assembled ratios were not altered by addition of nucleoside triphosphates, additional EGTA, or sulfhydryl protecting agents, and did not vary with preparation times of 30–90 min. The colchicine-binding reaction and decay of colchicine-binding activity with time were similar in extracts of different aged mouse brains, with neonatal slightly more stable than adult. Pools of tubulin from any age which were soluble at room temperature (unpolymerized) could not repolymerize well in vitro when incubated with GTP at 37 °C, whereas pools of tubulin which were sedimentable at room temperature (polymerized) could be redissolved at 0 °C and readily reassembled at 37 °C. The neonatal extract tubulin was thus more polymerization competent than the adult extracts; this correlates with a greater proportion of assembled tubulin in extracts at room temperature and possibly in vivo.     

Microtubule organization by cross-linking and bundling proteins.

To understand microtubule function the factors regulating their spatial organization and their interaction with cellular organelles, including other microtubules, must be elucidated. Many proteins are implicated in these organizational events and the known consequences of their actions within the cell are increasing. For example, the function of microtubule bundles at the surfaces of polarized cells has recently received attention, as has the action in cortical rotation of a transient arrangement of microtubules found beneath the vegetal surface of fertilized frog eggs. The in vivo association of microtubules during early Xenopus oogenesis has added interest as microtubules bundled in cell-free extracts are protected against the action of a severing protein found in this animal. A 52 kDa F-actin bundling protein purified from Physarum polycephalum organizes microtubules and causes the cobundling of microtubules and microfilaments. These observations, in concert with others that are presented, emphasize the diversity within the family of microtubule cross-linking proteins. The challenge is to determine which proteins are relevant from a physiological perspective, to ascertain their molecular mechanisms of action and to describe how they affect cytoplasmic organization and cell function. To realize this objective, the proteins which cross-link and bundle microtubules must be investigated by techniques which reveal different but related aspects of their properties. Cloning and sequencing of genes for cross-linking proteins, their subcellular localization especially as microtubule-related changes in cell morphology are occurring and the application of genetic studies are necessary. Study of the neural MAP provides the best example of just how powerful current experimental approaches are and at the same time shows their limits. The neural MAP have long been noted for their enhancement of tubulin assembly and microtubule stability. Their spatial distribution has been studied during the morphogenesis of neural cells. Sequencing of cloned genes has revealed the functional domains of neural MAP including carboxy-terminal microtubule-binding sites. Similarities to microtubule binding proteins from other cell types stimulate interest in the neural MAP and further suggest their importance in microtubule organization. For example, MAP4 enjoys a wide cellular distribution and has microtubule-binding sequences very similar to those in the neural MAP. Moreover, the nontubulin proteins of marginal bands are immunologically related to neural MAP, indicating shared structural/functional domains. Even with these findings the mechanism by which neural MAP cross-link microtubules remains uncertain. Indeed, some researchers express doubt that microtubule cross-linking is actually a function of neural MAP in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microtubule motors in spindle and chromosome motility.

Many of the kinesin microtubule motor proteins discovered during the past 8-9 years have roles in spindle assembly and function or chromosome movement during meiosis or mitosis. The discovery of kinesin motor proteins with a clear involvement in spindle and chromosome motility, together with recent evidence that cytoplasmic dynein plays a role in chromosome distribution, has attracted great interest. The identification of microtubule motors that function in chromosome distribution represents a major advance in understanding the forces that underlie chromosome and spindle movements during cell division.     

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.     

SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing

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Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

Microtubule cytoskeleton: No longer an also Ran.

The Ran GTPase cycle has been extensively studied in the context of nuclear transport. Recent work indicates that this GTPase cycle also plays an important role in regulating the microtubule cytoskeleton.