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1.
Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis. 总被引:16,自引:0,他引:16
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N Larsson U Marklund H M Gradin G Brattsand M Gullberg 《Molecular and cellular biology》1997,17(9):5530-5539
Oncoprotein 18 (Op18; also termed p19, 19K, metablastin, stathmin, and prosolin) is a conserved protein that regulates microtubule (MT) dynamics. Op18 is multisite phosphorylated on four Ser residues during mitosis; two of these Ser residues, Ser-25 and Ser-38, are targets for cyclin-dependent protein kinases (CDKs), and the other two Ser residues, Ser-16 and Ser-63, are targets for an unidentified protein kinase. Mutations of the two CDK sites have recently been shown to result in a mitotic block caused by destabilization of MTs. To understand the role of Op18 in regulation of MT dynamics during mitosis, in this study we dissected the functions of all four phosphorylation sites of Op18 by combining genetic, morphological, and biochemical analyses. The data show that all four phosphorylation sites are involved in switching off Op18 activity during mitosis, an event that appears to be essential for formation of the spindle during metaphase. However, the mechanisms by which specific sites down-regulate Op18 activity differ. Hence, dual phosphorylation on the CDK sites Ser-25 and Ser-38 appears to be required for phosphorylation of Ser-16 and Ser-63; however, by themselves, the CDK sites are of only minor importance in direct regulation of Op18 activity. Subsequent phosphorylation of either Ser-16, Ser-63, or both efficiently switches off Op18 activity. 相似文献
2.
Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of "mitotic chromatin." Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control. 相似文献
3.
Regulation of microtubule dynamics by Ca2+/calmodulin-dependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18. 总被引:10,自引:1,他引:9
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H Melander Gradin U Marklund N Larsson T A Chatila M Gullberg 《Molecular and cellular biology》1997,17(6):3459-3467
Oncoprotein 18 (Op18; also termed p19, 19K, p18, prosolin, and stathmin) is a regulator of microtubule (MT) dynamics and is phosphorylated by multiple kinase systems on four Ser residues. In addition to cell cycle-regulated phosphorylation, external signals induce phosphorylation of Op18 on Ser-25 by the mitogen-activated protein kinase and on Ser-16 by the Ca2+/calmodulin-dependent kinase IV/Gr (CaMK IV/Gr). Here we show that induced expression of a constitutively active mutant of CaMK IV/Gr results in phosphorylation of Op18 on Ser-16. In parallel, we also observed partial degradation of Op18 and a rapid increase of total cellular MTs. These results suggest a link between CaMK IV/Gr, Op18, and MT dynamics. To explore such a putative link, we optimized a genetic system that allowed conditional coexpression of a series of CaMK IV/Gr and Op18 derivatives. The result shows that CaMK IV/Gr can suppress the MT-regulating activity of Op18 by phosphorylation on Ser-16. In line with these results, by employing a chemical cross-linking protocol, it was shown that phosphorylation of Ser-16 is involved in weakening of the interactions between Op18 and tubulin. Taken together, these data suggest that the mechanism of CaMK IV/Gr-mediated suppression of Op18 activity involves both partial degradation of Op18 and direct modulation of the MT-destabilizing activity of this protein. These results show that Op18 phosphorylation by CaMK IV/Gr may couple alterations of MT dynamics in response to external signals that involve Ca2+. 相似文献
4.
Per Holmfeldt 《Experimental cell research》2010,316(12):2017-2026
Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18→E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability. 相似文献
5.
Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin 总被引:11,自引:4,他引:7
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Bonnie Howell Niklas Larsson Martin Gullberg Lynne Cassimeris 《Molecular biology of the cell》1999,10(1):105-118
Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (α, β)-methylene diphosphonate–tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis. 相似文献
6.
Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans 总被引:8,自引:3,他引:5
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《The Journal of cell biology》1984,99(3):830-838
We used immunofluorescent microscopy to characterize microtubule (MT) architecture in wild-type and mutant protoplasts of Aspergillus nidulans at interphase and at mitosis. Because the visualization of MTs by immunofluorescence is technically difficult in intact hyphae of A. nidulans, we developed a method for removing the cell wall under conditions that do not perturb cell physiology, as evidenced by the fact that the resulting protoplasts undergo nuclear division at a normal rate and that cell cycle mutant phenotypes are expressed at restrictive temperature. Interphase cells exhibited an extensive network of cytoplasmic MTs. During mitosis the cytoplasmic MTs mostly disappeared and an intranuclear mitotic spindle appeared. We have previously shown that the benA 33 beta-tubulin mutation causes hyperstabilization of the mitotic spindle, and we have presented additional indirect evidence that suggested that the tubA1 and tubA4 alpha-tubulin mutations destabilize spindle MTs. In this paper, we show that the benA33 mutation increases the stability of cytoplasmic MTs as well as spindle MTs and that the tubA1 and tubA4 mutations destabilize both spindle and cytoplasmic MTs. 相似文献
7.
Oncoprotein 18 is a phosphorylation-responsive regulator of microtubule dynamics. 总被引:15,自引:1,他引:14
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Oncoprotein 18 (Op18, also termed p19, p18, prosolin or stathmin) is a cytosolic protein of previously unknown function. Phosphorylation of Op18 is cell cycle regulated by cyclin-dependent kinases (CDKs), and expression of a 'CDK target site-deficient mutant' results in a phenotype indicative of a role for Op18 during mitosis. This phenotype is compatible with the idea that Op18 is a phosphorylation-responsive regulator of microtubule (MT) dynamics. Therefore, in this study, we analyzed MTs in cells induced to express either wild-type or mutated Op18. The results showed that wild-type Op18 and a CDK target site mutant both efficiently elicited rapid depolymerization of MTs. This result contrasts with clear-cut differences in their cell cycle phenotypes. Morphological analysis of MTs explained this apparent discrepancy: while interphase MTs were depolymerized in cells expressing either Op18 derivative, apparently normal mitotic spindles were formed only in cells overexpressing wild-type Op18. This result correlates with our finding that only mutated Op18 causes a block during mitosis. Hence, we conclude that Op18 decreases MT stability and that this activity of Op18 is subject to cell cycle regulation by CDKs. 相似文献
8.
Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo. Both a microtubule catastrophe-promoting activity and a tubulin sequestering activity were demonstrated for stathmin in vitro, and both could contribute to microtubule depolymerization in vivo. Stathmin activity can be turned down by extensive phosphorylation on its four phosphorylatable serines, and down-regulation of stathmin activity by phosphorylation is necessary for cells to proceed through mitosis. We show here that microinjection of a nonphosphorylatable Ser to Ala (4A) quadruple mutant in Xenopus two-cell stage embryos results in cell cleavage arrest in the injected blastomeres and aborted development, whereas injection of a pseudo-phosphorylated Ser to Glu quadruple mutant (4E) does not prevent normal development. Addition of these mutants to mitotic cytostatic factor-arrested extracts in which spindle assembly was induced led to a dramatic reduction of spindle size with 4A stathmin, and to a moderate increase with 4E stathmin, but both localized to spindle poles. Interestingly, the microtubule assembly-dependent phosphorylation of endogenous stathmin was abolished in the presence of 4A stathmin, but not of 4E stathmin. Altogether, this shows that the phosphorylation-mediated regulation of stathmin activity during the cell cycle is essential for early Xenopus embryonic development. 相似文献
9.
Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18. 相似文献
10.
N Larsson B Segerman B Howell K Fridell L Cassimeris M Gullberg 《The Journal of cell biology》1999,146(6):1289-1302
Oncoprotein18/stathmin (Op18) is a regulator of microtubule (MT) dynamics that binds tubulin heterodimers and destabilizes MTs by promoting catastrophes (i.e., transitions from growing to shrinking MTs). Here, we have performed a deletion analysis to mechanistically dissect Op18 with respect to (a) modulation of tubulin GTP hydrolysis and exchange, (b) tubulin binding in vitro, and (c) tubulin association and MT-regulating activities in intact cells. The data reveal distinct types of region-specific Op18 modulation of tubulin GTP metabolism, namely inhibition of nucleotide exchange and stimulation or inhibition of GTP hydrolysis. These regulatory activities are mediated via two-site cooperative binding to tubulin by multiple nonessential physically separated regions of Op18. In vitro analysis revealed that NH(2)- and COOH-terminal truncations of Op18 have opposite effects on the rates of tubulin GTP hydrolysis. Transfection of human leukemia cells with these two types of mutants result in similar decrease of MT content, which in both cases appeared independent of a simple tubulin sequestering mechanism. However, the NH(2)- and COOH-terminal-truncated Op18 mutants regulate MTs by distinct mechanisms as evidenced by morphological analysis of microinjected newt lung cells. Hence, mutant analysis shows that Op18 has the potential to regulate tubulin/MTs by more than one specific mechanism. 相似文献
11.
Mutations of oncoprotein 18/stathmin identify tubulin-directed regulatory activities distinct from tubulin association
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Larsson N Segerman B Gradin HM Wandzioch E Cassimeris L Gullberg M 《Molecular and cellular biology》1999,19(3):2242-2250
Oncoprotein 18/stathmin (Op18) is a recently identified phosphorylation-responsive regulator of the microtubule (MT) system. It was originally proposed that Op18 specifically regulates dynamic properties of MTs by associating with tubulin, but it has subsequently been proposed that Op18 acts simply by sequestering of tubulin heterodimers. We have dissected the mechanistic action of Op18 by generation of two distinct classes of mutants. One class has interruptions of the heptad repeats of a potential coiled-coil region of Op18, and the other involves substitution at all four phosphorylation sites with negatively charged Glu residues. Both types of mutation result in Op18 proteins with a limited decrease in tubulin complex formation. However, the MT-destabilizing activities of the coiled-coil mutants are more severely reduced in transfected leukemia cells than those of the Glu-substituted Op18 derivative, providing evidence for tubulin-directed regulatory activities distinct from tubulin complex formation. Analysis of Op18-mediated regulation of tubulin GTPase activity and taxol-promoted tubulin polymerization showed that while wild-type and Glu-substituted Op18 derivatives are active, the coiled-coil mutants are essentially inactive. This suggests that Op18-tubulin contact involves structural motifs that deliver a signal of regulatory importance to the MT system. 相似文献
12.
In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells 总被引:18,自引:5,他引:13
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《The Journal of cell biology》1994,124(1):129-137
Phosphorylation of the regulatory light chain of myosin II (MLC) controls the contractility of actomyosin in nonmuscle and muscle cells. It has been reported that cdc2 phosphorylates MLC in vitro at Ser-1 or Ser-2 and Thr-9 which protein kinase C phosphorylates (Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. K. Cisek, J. L. Corden, and T. D. Pollard. 1992 J. Cell Biol. 118:595-605). We have examined in vivo phosphorylation of MLC during mitosis and after the release of mitotic arrest. Phosphate incorporation of MLC in mitotic cells is found to be 6-12 times greater than that in nonmitotic cells. Phosphopeptide maps have revealed that the MLC from mitotic cells is phosphorylated at Ser-1 and/or Ser-2 (Ser-1/2), but not at Thr-9. MLC is also phosphorylated to a much lesser extent at Ser-19 which myosin light chain kinase phosphorylates. On the other hand, MLC of nonmitotic cells is phosphorylated at Ser-19 but not at Ser-1/2. The extent of phosphate incorporation is doubled at 30 min after the release of mitotic arrest when some cells start cytokinesis. Phosphopeptide analyses have revealed that the phosphorylation at Ser-19 is increased 20 times, while the phosphorylation at Ser-1/2 is decreased by half. This high extent of MLC phosphorylation at Ser-19 is maintained for another 30 min and gradually decreased to near the level of interphase cells as cells complete spreading at 180 min. On the other hand, phosphorylation at Ser-1/2 is decreased to 18% at 60 min, and is practically undetectable at 180 min after the release of mitotic arrest. The stoichiometry of MLC phosphorylation has been determined by quantitation of phosphorylated and unphosphorylated forms of MLC separated on 2D gels. The molar ratio of phosphorylated MLC to total MLC is found to be 0.16 +/- 0.06 and 0.31 +/- 0.05 in interphase and mitotic cells, respectively. The ratio is increased to 0.49 +/- 0.05 at 30 min after the release of mitotic arrest. These results suggest that the change in the phosphorylation site from Ser-1/2 to Ser-19 plays an important role in signaling cytokinesis. 相似文献
13.
Regulation of Microtubule Dynamics by Extracellular Signals: cAMP-dependent Protein Kinase Switches Off the Activity of Oncoprotein 18 in Intact Cells 总被引:12,自引:0,他引:12
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Helena Melander Gradin Niklas Larsson Ulrica Marklund Martin Gullberg 《The Journal of cell biology》1998,140(1):131-141
Oncoprotein 18 (Op18, also termed p19, 19K, metablastin, stathmin, and prosolin) is a recently identified regulator of microtubule (MT) dynamics. Op18 is a target for both cell cycle and cell surface receptor-coupled kinase systems, and phosphorylation of Op18 on specific combinations of sites has been shown to switch off its MT-destabilizing activity. Here we show that induced expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) results in a dramatic increase in cellular MT polymer content concomitant with phosphorylation and partial degradation of Op18. That PKA may regulate the MT system by downregulation of Op18 activity was evaluated by a genetic system allowing conditional co-expression of PKA and a series of kinase target site–deficient mutants of Op18. The results show that phosphorylation of Op18 on two specific sites, Ser-16 and Ser-63, is necessary and sufficient for PKA to switch off Op18 activity in intact cells. The regulatory importance of dual phosphorylation on Ser-16 and Ser-63 of Op18 was reproduced by in vitro assays. These results suggest a simple model where PKA phosphorylation downregulates the MT-destabilizing activity of Op18, which in turn promotes increased tubulin polymerization. Hence, the present study shows that Op18 has the potential to regulate the MT system in response to external signals such as cAMP-linked agonists. 相似文献
14.
Deciphering the cellular functions of the Op18/Stathmin family of microtubule-regulators by plasma membrane-targeted localization
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The Op18/stathmin family of microtubule regulators includes the ubiquitous cytosolic Op18/stathmin (Op18) and the neuronal, primarily Golgi-associated proteins SCG10 and RB3, which all form ternary complexes with two head-to-tail-aligned tubulin heterodimers. To understand the physiological significance of previously observed differences in ternary complex stability, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. Herein, we show that, in contrast to constitutively active CD2-Op18-tetraA, both the CD2-SCG10 and CD2-RB3 chimeras sequestered tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels and consequent delayed formation of mitotic spindles. However, all three CD2 chimeras, including the tubulin sequestration-incompetent CD2-Op18-tetraA, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during interphase, but not during mitosis, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. However, the differences in tubulin binding observed in cells also indicate conceptual differences between the functions of low-abundance neural family members, which will accumulate tubulin at specific cellular compartments, and the abundant cytosolic Op18 protein, which will not. 相似文献
15.
Aneugenic activity of Op18/stathmin is potentiated by the somatic Q18-->e mutation in leukemic cells
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Op18/stathmin (Op18) is a phosphorylation-regulated microtubule destabilizer that is frequently overexpressed in tumors. The importance of Op18 in malignancy was recently suggested by identification of a somatic Q18-->E mutation of Op18 in an adenocarcinoma. We addressed the functional consequences of aberrant Op18 expression in leukemias by analyzing the cell cycle of K562 cells either depleted of Op18 by expression of interfering hairpin RNA or induced to express wild-type or Q18E substituted Op18. We show here that although Op18 depletion increases microtubule density during interphase, the density of mitotic spindles is essentially unaltered and cells divide normally. This is consistent with phosphorylation-inactivation of Op18 during mitosis. Overexpression of wild-type Op18 results in aneugenic activities, manifest as aberrant mitosis, polyploidization, and chromosome loss. One particularly significant finding was that the aneugenic activity of Op18 was dramatically increased by the Q18-->E mutation. The hyperactivity of mutant Op18 is apparent in its unphosphorylated state, and this mutation also suppresses phosphorylation-inactivation of the microtubule-destabilizing activity of Op18 without any apparent effect on its phosphorylation status. Thus, although Op18 is dispensable for mitosis, the hyperactive Q18-->E mutant, or overexpressed wild-type Op18, exerts aneugenic effects that are likely to contribute to chromosomal instability in tumors. 相似文献
16.
Andersen SS 《Trends in cell biology》2000,10(7):261-267
The way that microtubules reorganize from their long, stable interphase configuration to form the mitotic spindle remains a challenging and unsolved question. It is now widely recognized that microtubule polymerization during the cell cycle is regulated by a balance between microtubule-stabilizing and-destabilizing factors. Stabilizing factors include a large group of microtubule-associated proteins (MAPs; e.g. MAP4, XMAP215, XMAP230/XMAP4 and XMAP310) and the destabilizing factors are a growing family of proteins (e.g. Stathmin/Op18 and XKCM1). Recent studies have allowed a mechanistic dissection of how these stabilizing and destabilizing factors regulate microtubule dynamics and spindle assembly. 相似文献
17.
The oncoprotein 18/stathmin family of microtubule destabilizers. 总被引:19,自引:0,他引:19
Lynne Cassimeris 《Current opinion in cell biology》2002,14(1):18-24
The past several years have seen major advances in our understanding of the mechanisms of microtubule destabilization by oncoprotein18/stathmin (Op18/stathmin) and related proteins. New structural information has clearly shown how members of the Op18/stathmin protein family bind tubulin dimers and suggests models for how these proteins stimulate catastrophe, the transition from microtubule growth to shortening. Regulation of Op18/stathmin by phosphorylation continues to capture much attention. Studies suggest that phosphorylation occurs in a localized fashion, resulting in decreased microtubule destabilizing activity near chromatin or microtubule polymer. A spatial gradient of inactive Op18/stathmin associated with chromatin or microtubules could contribute significantly to mitotic spindle assembly. 相似文献
18.
Sebastian Mana-Capelli Janel R. McLean Chun-Ti Chen Kathleen L. Gould Dannel McCollum 《Molecular biology of the cell》2012,23(23):4592-4600
In Schizosaccharomyces pombe, a late mitotic kinase pathway called the septation initiation network (SIN) triggers cytokinesis. Here we show that the SIN is also involved in regulating anaphase spindle elongation and telophase nuclear positioning via inhibition of Klp2, a minus end–directed kinesin-14. Klp2 is known to localize to microtubules (MTs) and have roles in interphase nuclear positioning, mitotic chromosome alignment, and nuclear migration during karyogamy (nuclear fusion during mating). We observe SIN-dependent disappearance of Klp2 from MTs in anaphase, and we find that this is mediated by direct phosphorylation of Klp2 by the SIN kinase Sid2, which abrogates loading of Klp2 onto MTs by inhibiting its interaction with Mal3 (EB1 homologue). Disruption of Klp2 MT localization is required for efficient anaphase spindle elongation. Furthermore, when cytokinesis is delayed, SIN inhibition of Klp2 acts in concert with microtubules emanating from the equatorial microtubule-organizing center to position the nuclei away from the cell division site. These results reveal novel functions of the SIN in regulating the MT cytoskeleton and suggest that the SIN may have broader functions in regulating cellular organization in late mitosis than previously realized. 相似文献
19.
Summary To examine the effects exerted on the microtubule (MT) cytoskeleton by dinitrophenol/deoxyglucose (DNP/DOG) and nocodazole, live PtK1 cells were treated with the drugs and then fixed and examined by immunofluorescence staining and electronmicroscopy. DNP/DOG had little effect on interphase MTs. In mitotic cells, kinetochore and some astral fibers were clearly shortened in metaphase figures by DNP/DOG. Nocodazole rapidly broke down spindle MTs (except those in the midbody), while interphase cells showed considerable variation in the susceptibility of their MTs. Nocodazole had little effect on MTs in energy-depleted (DNP/DOG-treated) cells. When cytoplasmic MTs had all been broken down by prolonged nocodazole treatment and the cells then released from the nocodazole block into DNP/DOG, some MT reassembly occurred in the ATP-depleted state. MTs in permeabilized, extracted cells were also examined with antitubulin staining; the well-preserved interphase and mitotic arrays of MTs showed no susceptibility to nocodazole. In contrast, MTs suffered considerable breakdown by ATP, GTP and ATPS; AMPPNP had little effect. This susceptibility of extracted MT cytoskeleton to nucleotide phosphates was highly variable; some interphase cells lost all MTs, most were severely affected, but some retained extensive MT networks; mitotic spindles were diminished but structurally coherent and more stable than most interphase MT arrays.We suggest that: 1. in the living cell, ATP or nucleotide triphosphates (NTPs) are necessary for normal and nocodazole-induced MT disassembly; 2. the NTP requirement may be for phosphorylation; 3. shortening of kinetochore fibers may be modulated by compression and require ATP; 4. many of these results cannot be accomodated by the dynamic equilibrium theory of MT assembly/disassembly; 5. the use and role of ATP on isolated spindles may have to be reevaluated due to the effects ATP has on the spindle cytoskeleton of permeabilized cells. 相似文献
20.
Indirect immunofluorescence of microtubules in Dictyostelium discoideum. A study with polyclonal and monoclonal antibodies to tubulins 总被引:1,自引:0,他引:1
Amebae of D. discoideum on coverslips were fixed in situ with glutaraldehyde and permeabilized with Triton X-100. Of six antibodies tested, only a monoclonal antibody to yeast tubulin consistently gave bright fluorescence. Counterstaining with DAPI facilitated the identification of interphase and mitotic stages. Most microtubules (MTs) in interphase amebae emanated from a nucleus-associated centre that had a non-fluorescent core. Amebae in early stages of mitosis lacked cytoplasmic MTs almost entirely. The nascent spindle in prophase appeared as a brightly fluorescent dot, whereas the prometaphase spindle was a short rod. Spindles in metaphase and anaphase nuclei were more elongate, some consisting of several fluorescent lines. Astral MTs were prominent on spindles in anaphase and telophase. Asters are obviously converted to the interphase complex of MTs in post-mitotic cells, while the shaft-like remnant of the central spindle disappears. The cyclical changes in the MT system related to cell division resemble those observed in higher eukaryotes and probably reflect changes in the locomotory behavior of the amebae rather than changes in cell shape. 相似文献