首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
Microtubules in ascidian eggs during meiosis, fertilization, and mitosis   总被引:14,自引:0,他引:14  
The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic crescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.  相似文献   

2.
Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.  相似文献   

3.
Cells have developed diverse ways to separate two microtubule asters to form a mitotic spindle. Here, I focus on two mechanisms used to position asters around chromosomes during mitosis: first, aster migration around the nuclear envelope and, second, aster attachment to a contractile cortex at the plasma membrane after the nuclear envelope has broken down. Although certain cell types use one mechanism predominantly, most rely on both to ensure proper spindle assembly.  相似文献   

4.
Structural constituents of the spindle apparatus essential for cleavage induction remain undefined. Findings from various cell types using different approaches suggest the importance of all structural constituents, including asters, the central spindle, and chromosomes. In this study, we systematically dissected the role of each constituent in cleavage induction in grasshopper spermatocytes and narrowed the essential one down to bundled microtubules. Using micromanipulation, we produced "cells" containing only asters, a truncated central spindle lacking both asters and chromosomes, or microtubules alone. We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present. Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in "metaphase," disassembling in "anaphase," and bundling into arrays in "telophase." Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress. We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.  相似文献   

5.
We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.  相似文献   

6.
NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.  相似文献   

7.
Previous immunofluorescence studies of microtubule distribution in fertilized sea urchin eggs have suffered from poor resolution caused by cell thickness, unavoidable artifacts resulting from excessive flattening, or extraction by detergents of membranes and other lipid-containing structures that may be of interest in relation to the microtubules. To avoid these difficulties, we have developed a fixation and embedding protocol based on buffered paraformaldehyde fixation and butyl-methyl methacrylate embedment, which allows immunofluorescence staining of 0.5-1 micron sections. Polymerization artifacts are reduced by polymerizing the methacrylate at a relatively low temperature (40-45 degrees C) and by flat embedding for more uniform polymerization. Using this method, we have examined mitotic stages in the first cleavage cycle of the sea urchin Strongylocentrotus purpuratus. We provide evidence that the interphase microtubules that appear after first division are not derived from the mitotic asters but are new structures growing from organizing centers within the degenerating mitotic asters. During the transition from mitosis to interphase, there is a temporary overlap of old and new microtubules to form a very large composite aster at telophase before the old structure finally disappears.  相似文献   

8.
《The Journal of cell biology》1994,126(6):1509-1526
MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114- kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically. We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.  相似文献   

9.
Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.  相似文献   

10.
Mitosis and microtubule organizational changes in rice root-tip cells   总被引:1,自引:0,他引:1  
The pattern of change of the microtubule cytoskeleton of the root-tip cells of rice during mitosis was studied using immunofluorescence technic and confocal laser scanning microscopy. All the major stages of ceil division including preprophase, prophase, metaphase, anaphase and telophase were observed. The most significant finding was that in the preprophase cells microtubules radiating from the nuclear surface to the cortex were frequently seen. During development these microtubules became closely associated with the preprophase band and prophase spindie indicating that the microtubules radiating from the nuclear surface, the preprophase band and the prophazc spindle were structurally and functionally closely related to each other. Granule-like anchorage sites for the radiating microtubules at the muclear surface were often seen and the possibility that these gramle-like anchorage sites might represent the microtubule organizing centres was discussed.  相似文献   

11.
We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.  相似文献   

12.
NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.  相似文献   

13.
The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.  相似文献   

14.
The organization of microtubules within the surface caps of Drosophila embryos is described for the mitotic cycles of the syncytial blastoderm stage (particularly cycle 10), and for the subsequent cellularization process. Tubulin was labelled with the well characterized monoclonal antibody YL 1/2 (Kilmartin et al., J cell biol 93 (1982) 576). Each surface cap was found to contain an array of microtubules running around the nucleus. The microtubules originated at prominent centrosomes located close to the apical surface of each cap nucleus. During mitosis the spindle microtubules stained strongly for tubulin. A novel finding was that the spindle microtubules of the interzone region appeared to reduce their connections with the centrosomes at the end of anaphase. The spindle remnant remained in position during telophase but then became smaller in size, disappearing by interphase. At this phase of the cell cycle duplication of the aster centrosomes occurred. The cellular blastoderm stage was marked by a change in the main axis of microtubule orientation. The centrosomes of each cap separated somewhat and formed initiation centres for the development of a well developed basket of microtubules around each nucleus, but now perpendicular to the surface. The microtubule baskets were seen to extend in parallel with nuclear elongation, but not in concert with growth of the cell membranes, which extended some way beneath the bases of the nuclei.  相似文献   

15.
Influence of M-phase chromatin on the anisotropy of microtubule asters   总被引:2,自引:1,他引:1  
In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long- range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long- range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.  相似文献   

16.
During mitosis, rebuilding of the nucleolus is a step-wise process that, above all, includes an assembly of nucleolus-derived foci (NDF) in the cytoplasm of telophase cells. In this study, we performed a comparative analysis of NDF formation in mitotic cells of various mammalian cell cultures, such as green monkey CV1 cells, human HeLa cells, mouse 3T3 cells, and pig PK cells, both in control and following inhibition of rRNA synthesis by actinomycin D or by an adenosine analogue, DRB. The results obtained show that in all examined cell types NDF are formed shortly after or simultaneously with the onset of chromosome segregation to the poles of the mitotic spindle. However, an efficiency of NDF assembly, i.e. the number of NDF per anaphase or telophase cell, and the portion of anaphase and telophase cells containing NDF vary in different cell cultures, being most prominent in CVI and HeLa cells. In these cells, the vast majority of NDF accumulate several proteins of the mature nucleolus, such as B23/nucleophopmin, C23/nucleotin, fibrillarin, and, to a lesser extent, Nop52. The rRNA harbored by NDF is synthesized several hours prior mitosis, and plays an essential role the maintenance of NDF structural integrity. Starting from early stages of the assembly onwards, the NDF are predominantly located in the area occupied by aster microtubules of the mitotic spindle.  相似文献   

17.
Taxol, a microtubule stabilizing agent, has been used to study changes in spindle microtubule organization during mitosis. PtK1 cells have been treated with 5 μg/ml taxol for brief periods to determine its effect on spindle architecture. During prophase taxol induces microtubules to aggregate, particularly evident in the region between the nucleus and cell periphery. Taxol induces astral microtubule formation in prometaphase and metaphase cells concomitant with a reduction in spindle length. At anaphase taxol induces an increase in length in astral microtubules and reduces microtubule length in the interzone. Taxol-treated telophase cells show a reduction in the rate of furrowing and astral microtubules lack a discrete focus and are arranged more diffusely on the surface of the nuclear envelope. In summary, taxol treatment of cells prior to anaphase produces an increase in astral microtubules, a reduction in kinetochore microtubules and a decrease in spindle length. Brief taxol treatments during anaphase through early G1 promotes stabilization of microtubules, an increase in the length of astral microtubules and a delayed rate of cytokinesis.  相似文献   

18.
Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.  相似文献   

19.
HeLa cells blocked in metaphase with 0.04 micrograms/ml of the microtubule poison nocodazole were shown to contain large numbers of microtubules with typical mitotic organization but no cenriole. Lysis of nocodazole-poisoned cells in a microtubule reassembly buffer containing 0.5 M PIPES, 2.5% dimethyl sulfoxide, 1 mM EDTA, 1 mM MgCl2, 1 mM GTP, 1% Triton X-165, 0.5% sodium deoxycholate, 0.2% SDS, pH 6.9, preserved metaphase aster structures 5 micrograms in diameter surrounded only by a thin, fibrous cell remnant. Inclusion of 2 mg/ml porcine brain microtubule protein in the lysis buffer produced asters up to 20 micrometers in diameter with a birefringent retardation of 5-6 nm. In these large asters the central microtubules had normal morphology, but peripheral microtubules were clearly abnormal. Our interpretation is that in high PIPES lysis buffer, exogenous brain tubulin adds to the distal ends of preexisting aster microtubules to form abnormal microtubules. This observation supports the assumptions made by Borisy and by Summers and Kirschner in their interpretation of growth experiments to determine the microtubule polarity in mitotic structures.  相似文献   

20.
Non-erythroid protein 4.1R (4.1R) consists of a complex family of isoforms. We have shown that 4.1R isoforms localize at the mitotic spindle/spindle poles and associate in a complex with the mitotic-spindle organization proteins Nuclear Mitotic Apparatus protein (NuMA), dynein, and dynactin. We addressed the mitotic function of 4.1R by investigating its association with microtubules, the main component of the mitotic spindles, and its role in mitotic aster assembly in vitro. 4.1R appears to partially co-localize with microtubules throughout the mitotic stages of the cell cycle. In vitro sedimentation assays showed that 4.1R isoforms directly interact with microtubules. Glutathione S-transferase (GST) pull-down assays using GST-4.1R fusions and mitotic cell extracts further showed that the association of 4.1R with tubulin results from both the membrane-binding domain and C-terminal domain of 4.1R. Moreover, 4.1R, but not actin, is a mitotic microtubule-associated protein; 4.1R associates with microtubules in the microtubule pellet of the mitotic asters assembled in mammalian cell-free mitotic extract. The organization of microtubules into asters depends on 4.1R in that immunodepletion of 4.1R from the extract resulted in randomly dispersed microtubules. Furthermore, adding a 135-kDa recombinant 4.1R reconstituted the mitotic asters. Finally, we demonstrated that a mitotic 4.1R isoform appears to form a complex in vivo with tubulin and NuMA in highly synchronized mitotic HeLa extracts. Our results suggest that a 135-kDa non-erythroid 4.1R is important to cell division, because it participates in the formation of mitotic spindles and spindle poles through its interaction with mitotic microtubules.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号