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1.
LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-alpha tubulin construct and a cell line permanently expressing GFP-alpha tubulin was established (LLCPK-1alpha). The mitotic index and doubling time for LLCPK-1alpha were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1alpha cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1alpha and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1alpha cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.  相似文献   

2.
Microtubule dynamics and tubulin interacting proteins   总被引:11,自引:0,他引:11  
Microtubule dynamics are crucial in generation of the mitotic spindle. During the transition from interphase to mitosis, there is an increase in the frequency of microtubule catastrophes. Recent work has identified two proteins, Op 18/stathmin and XKCM1, which can promote microtubule catastrophes in vitro and in cells or extracts. Although both of these proteins share the ability to bind tubulin dimers, their mechanisms of action in destabilizing microtubules are distinct.  相似文献   

3.
The transition from interphase to mitosis is marked by a dramatic change in microtubule dynamics resulting in the reorganization of the microtubule network that culminates in mitotic spindle formation. While the molecular basis for this change in microtubule organization remains obscure, it is currently thought that a balance in the activity of microtubule stabilizing and destabilizing factors regulates how dynamic cellular microtubules are. By mixing the microtubule stabilizer XMAP215 and the microtubule destabilizer XKCM1, reconstitution of in vivo-like microtubule dynamics has now been achieved in vitro.  相似文献   

4.
The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.  相似文献   

5.
Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-alpha-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.  相似文献   

6.
Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.  相似文献   

7.
The role of stathmin in the regulation of the cell cycle   总被引:24,自引:0,他引:24  
Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.  相似文献   

8.
We used high-resolution video microscopy to visualize microtubule dynamic instability in extracts of interphase sea urchin eggs and to analyze the changes that occur upon addition of 0.8-2.5 microM okadaic acid, an inhibitor of phosphatase 1 and 2A (PP1, PP2a) (Bialojan, D., and A. Takai. 1988. Biochem. J. 256:283-290). Microtubule plus-ends in these extracts oscillated between the elongation and shortening phases of dynamic instability at frequencies typical for interphase cells. Switching from elongation to shortening (catastrophe) was frequent, but microtubules persisted and grew long because of frequent switching back to elongation (rescue). Addition of okadaic acid to the extract induced rapid (< 5 min) conversion to short, dynamic microtubules typical of mitosis. The frequency of catastrophe doubled and the velocities of elongation and shortening increased slightly; however, the major change was an elimination of rescue. Thus, modulation of the rescue frequency by phosphorylation-dependent mechanisms may be a major regulatory pathway for selectively controlling microtubule dynamics without dramatically changing velocities of microtubule elongation and shortening.  相似文献   

9.
To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possible through regulation of microtubule dynamics in plant cells.  相似文献   

10.
Microtubules are dynamic polymers that participate in multiple cellular processes such as vesicular transport and cell division. Microtubule dynamics alter dramatically during the cell cycle. An excellent system to study microtubule dynamics is Xenopus egg extracts since it is a system that is open to manipulation. The extracts can be cycled between mitosis and interphase allowing the study of microtubules in these phases as well as during cell cycle transitions. Here, we provide simple assays to study microtubules in extracts and in vitro using purified components. Protocols are provided for the purification of frog tubulin, microtubule pelleting from extracts and in vitro, assembly of microtubule structures in extracts, and isolation of microtubule-associated proteins from extract. These methods can be used to analyze the effect of a protein of interest on the microtubule cytoskeleton.  相似文献   

11.
《The Journal of cell biology》1994,127(5):1289-1299
The reorganization from a radial [corrected] interphase microtubule (MT) network into a bipolar spindle at the onset of mitosis involves a dramatic change in MT dynamics. Microtubule-associated proteins (MAPs) and other factors are thought to regulate MT dynamics both in interphase and in mitosis. In this study we report the purification and functional in vitro characterization of a 230-KD MAP from Xenopus egg extract (XMAP230). This protein is present in eggs, oocytes, testis and a Xenopus tissue culture cell line. It is apparently absent from non- dividing cells in which an immunologically related 200-kD protein is found. XMAP230 is composed of two isoforms with slightly different molecular masses and pIs. It is localized to interphase MTs, dissociates from MTs at the onset of prophase and specifically binds to spindle MTs during metaphase and anaphase. The dissociation constant of XMAP230 is 500 nM, the stoichiometry of binding to MTs is between 1:8 and 1:4, and the in vivo concentration is approximately 200 nM. Both isoforms are phosphorylated and have reduced affinity for microtubules in mitotic extracts. Analysis of the effect of XMAP230 on MT dynamics by video microscopy shows that it increases the growth rate, decreases the shrinking rate of MTs and strongly suppresses catastrophes. These results suggest that in vivo, XMAP230 participates in the control of the MT elongation rate, stabilizes MTs and locally modulates MT dynamics during mitosis.  相似文献   

12.
Entry into mitosis depends on the activity of cyclin‐dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55δ subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle—high in interphase and suppressed during mitosis. Depletion of PP2A–B55δ (in interphase) from ‘cycling’ frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A–B55δ was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A–B55δ in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.  相似文献   

13.
14.
Importin beta is a mitotic target of the small GTPase Ran in spindle assembly   总被引:23,自引:0,他引:23  
The GTPase Ran has recently been shown to stimulate microtubule polymerization in mitotic extracts, but its mode of action is not understood. Here we show that the mitotic role of Ran is largely mediated by the nuclear transport factor importin beta. Importin beta inhibits spindle formation in vitro and in vivo and sequesters an aster promoting activity (APA) that consists of multiple, independent factors. One component of APA is the microtubule-associated protein NuMA. NuMA and other APA components are discharged from importin beta by RanGTP and induce spindle-like structures in the absence of centrosomes, chromatin, or Ran. We propose that RanGTP functions in mitosis as in interphase by locally releasing cargoes from transport factors. In mitosis, this promotes spindle assembly by organizing microtubules in the vicinity of chromosomes.  相似文献   

15.
Assembly of a mitotic spindle requires the accurate regulation of microtubule dynamics which is accomplished, at least in part, by phosphorylation-dephosphorylation reactions. Here we have investigated the role of serine-threonine phosphatases in the control of microtubule dynamics using specific inhibitors in Xenopus egg extracts. Type 2A phosphatases are required to maintain the short steady-state length of microtubules in mitosis by regulating the level of microtubule catastrophes, in part by controlling the the microtubule-destabilizing activity and phosphorylation of Op18/stathmin. Type 1 phosphatases are only required for control of microtubule dynamics during the transitions into and out of mitosis. Thus, although both type 2A and type 1 phosphatases are involved in the regulation of microtubule dynamics, they have distinct, non-overlapping roles.  相似文献   

16.
Microtubule-associated protein 2 (MAP2) derivatized with iodoacetamidotetramethylrhodamine or with iodoacetamidofluorescein binds to microtubules after injection into living interphase cells [Scherson et al, 1984]. The binding of derivatized MAP2 stabilized microtubules in vitro; it was therefore important to check if the binding of MAP2 in vivo perturbed the dynamics and organization of the microtubule network. We have addressed these questions by studying the effect of the injection of derivatized MAP2 on mitosis in PtK 1 cells and on the recovery of the microtubule network from low temperature incubation in interphase cells. We found that the presence of derivatized MAP2 did not change the duration of any mitotic stage and that the injected cell normally completed mitosis. We subsequently showed that the injected MAP2 bound to the microtubules within 5 minutes after injection and remained bound throughout the course of mitosis. The reorganization of the microtubule network upon cooling and rewarming was studied in the cytoplasm of human foreskin fibroblasts (356 cells). During the recovery, the distribution of the fluorescent MAP2 in living cells was identical with the microtubule pattern visualized by immunofluorescence in lysed and fixed cells. In these experiments, the fluorescent MAP2 bound to microtubules can be considered as a nonperturbing reporter of the microtubule network. This result is discussed in terms of the role of MAPs in the dynamics and organization of microtubules in living cells.  相似文献   

17.
The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.  相似文献   

18.
Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.  相似文献   

19.
R D Vale 《Cell》1991,64(4):827-839
Eukaryotic cells disassemble and reorganize their cytoskeleton during the cell cycle and in response to environmental cues. Disassembly of the actin cytoskeleton is aided by proteins that sever filamentous actin, but microtubule-severing proteins thus far have not been identified. Here, we describe an activity in extracts from Xenopus eggs that rapidly severs stable microtubules along their length. Severing is elicited by a protein(s) whose activity is greatly stimulated during mitosis through a posttranslational mechanism. The microtubule-severing factor may be involved in disassembling the interphase microtubule network prior to constructing the mitotic spindle.  相似文献   

20.
The nuclear-centrosome complex was isolated from interphase Chinese hamster ovary (CHO) cells, and, with exogenous brain tubulin as a source of subunits, the centrosome, while attached to the nucleus, was demonstrated to nucleate microtubule formation in vitro. We attempted to quantitate the nucleating activity in order to compare the activity of mitotic and interphase centrosomes. However, the proximity of the nucleus hindered these attempts, and efforts to chemically or mechanically remove the centrosome led to diminished nucleating activity. Therefore, the nuclear-centrosome complex was dissociated biologically through use of the cytochalasin B procedure for enucleation of cells. Cytoplasts were prepared that retained the centrosome. Lysis of the cytoplasts released free centrosomes that could nucleate microtubules in vitro. The nucleating activities of interphase and mitotic centrosomes were compared. In addition, through the use of whole-mount electron microscopy, the configuration of the centrioles was analyzed and the number of microtubules nucleated was determined as a function of the centriole cycle. Nucleating activity did not change discernibly throughout interphase but increased approximately fivefold at the transition to mitosis. Thus, we conclude that the nucleating activity of the centrosome is relatively independent of the centriole cycle but coupled to the mitotic cycle.  相似文献   

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