Stimulation of Microtubule Aster Formation and Spindle Assembly by the Small GTPase Ran

Wilde, Andrew; Zheng, Yixian

doi:10.1126/science.284.5418.1359

Cited by 371 publications

(318 citation statements)

References 23 publications

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“…Thus, although formation of Ran asters requires ␥ tubulin (Wilde and Zheng, 1999), it seems to be less dependent on pericentrin than does centrosome-mediated aster assembly.…”

Section: The C Terminus Of Pericentrin Disassembles Mitotic Asters Anmentioning

confidence: 94%

“…Treated nuclei were prefixed in 5% formaldehyde, spun onto coverslips through a 20% sucrose cushion by using a JS13.1 rotor at 8000 rpm 15 min, and postfixed in methanol (Ϫ20°C) before staining for immunofluorescence. Ran-mediated asters were prepared using constitutively active RanL43E as described previously (Wilde and Zheng, 1999). Centrosome-dependent and -independent Xenopus mitotic asters were fixed in formaldehyde on coverslips as described previously (Murray, 1991;Wilde and Zheng, 1999).…”

Section: Xenopus Extractsmentioning

confidence: 99%

“…Ran-mediated asters were prepared using constitutively active RanL43E as described previously (Wilde and Zheng, 1999). Centrosome-dependent and -independent Xenopus mitotic asters were fixed in formaldehyde on coverslips as described previously (Murray, 1991;Wilde and Zheng, 1999). …”

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confidence: 99%

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Mitosis-specific Anchoring of γ Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry

Zimmerman

Sillibourne

Rosa

et al. 2004

MBoC

274

258

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Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by ␥ tubulin ring complexes (␥ 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 ␥ TuRCs at spindle poles through an interaction with ␥ tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted ␥ tubulin localization and spindle organization in mitosis but had no effect on ␥ tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-␥ TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled ␥ 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 ␥ 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. INTRODUCTIONThe centrosome is the primary microtubule-organizing center in animal cells. At the centrosome core is a pair of barrel-shaped microtubule assemblies, the centrioles (Doxsey, 2001). Centrioles are capable of self-assembly (Marshall et al., 2001;Khodjakov et al., 2002) and can serve as templates for recruitment and organization of the surrounding pericentriolar matrix (Bobinnec et al., 1998;Kirkham et al., 2003). The pericentriolar material or centrosome matrix contains a high proportion of coiled coil proteins and is the site of microtubule nucleation. Within the matrix are large protein complexes of ␥ tubulin and associated proteins that have a ring-like structure and mediate the nucleation of microtubules called ␥ tubulin ring complexes or ␥ TuRCs (Moritz et al., 1995a;Zheng et al., 1995). Other proteins may share the ability to nucleate microtubules because centrosomes can organize microtubules in the absence of functional ␥ tubulin (Sampaio et al., 2001;Strome et al., 2001;Hannak et al., 2002).During cell cycle progression, centrosomes "mature" by recruiting additional ␥ TuRCs and several other proteins, resulting in an increase in the nucleation capacity of the centrosome (reviewed in Blagden and Glover, 2003). However, we still know very little about proteins that directly anchor ␥ TuRCs to centrosomes in vertebrate cells. ...

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“…Thus, although formation of Ran asters requires ␥ tubulin (Wilde and Zheng, 1999), it seems to be less dependent on pericentrin than does centrosome-mediated aster assembly.…”

Section: The C Terminus Of Pericentrin Disassembles Mitotic Asters Anmentioning

confidence: 94%

Section: Xenopus Extractsmentioning

confidence: 99%

See 1 more Smart Citation

Mitosis-specific Anchoring of γ Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry

Zimmerman

Sillibourne

Rosa

et al. 2004

MBoC

274

258

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“…Such a role of DdCP224 is plausible because, in Xenopus egg extracts, XMAP215 is known to be essential for self-organization of MTOCs induced by the small GTPase Ran (Ohba et al, 1999;Wilde and Zheng, 1999).…”

Section: Origin and Replication Of Supernumerary Centrosomesmentioning

confidence: 99%

Regulated Expression of the Centrosomal Protein DdCP224 Affects Microtubule Dynamics and Reveals Mechanisms for the Control of Supernumerary Centrosome Number

Gräf

Euteneuer

et al. 2003

MBoC

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The Dictyostelium XMAP215 family member DdCP224 is involved in centrosome duplication and cytokinesis and is concentrated at the centrosome and microtubule tips. Herein, we have created a DdCP224 promoter replacement mutant that allows both over-and underexpression. Overexpression led to supernumerary microtubule-organizing centers and, independently, an increase of the number of multinuclear cells. Electron microscopy demonstrated that supernumerary microtubule-organizing centers represented bona fide centrosomes. Live cell imaging of DdCP224-green fluorescent protein mutants also expressing green fluorescent protein-histone2B as a DNA label revealed that supernumerary centrosomes were also competent of cell cycle-dependent duplication. In contrast, underexpression of DdCP224 inhibited cell growth, reduced the number and length of astral microtubules, and caused nocodazole hypersensitivity. Moreover, microtubule regrowth after nocodazole removal was dependent on DdCP224. Underexpression also resulted in a striking disappearance of supernumerary centrosomes and multinuclear cells caused by previous overexpression. We show for the first time by live cell observation that the number of supernumerary centrosomes can be reduced either by centrosome fusion (coalescence) or by the formation of cytoplasts containing supernumerary centrosomes during cytokinesis. INTRODUCTIONIn dividing cells, control of centrosome number is essential for the fidelity of mitosis and maintenance of euploidy. Supernumerary centrosomes are a hallmark of tumor cells, although it is still a matter of discussion whether their appearance is a cause or a consequence of carcinogenesis (Lingle et al., 2002;Nigg, 2002). However, it is widely accepted that supernumerary centrosomes contribute to the formation of multipolar spindles and thus to defective chromosome segregation. Although most of the daughter cells resulting from such mitotic chaos will die, some of them will acquire the potential for neoplastic growth in spite of supernumerary centrosomes and aneuploidy, as they regain the ability to form a bipolar spindle. Brinkley (2001) suggested two mechanisms how this can be achieved: first, "deamplification" of supernumerary centrosomes by elimination or inactivation, and second, fusion or "coalescence" of supernumerary centrosomes resulting in one or two compound microtubule-organizing centers (MTOCs).Herein, we have studied the fate of supernumerary centrosomes in a simple model system. In Dictyostelium amoebae, one molecular component involved in the formation of supernumerary centrosomes is DdCP224, a member of the XMAP215 family of microtubule-associated proteins (Gräf et al., 2000). In most species, these proteins are long, thin, monomeric molecules with a size of ϳ 230 kDa (Cassimeris et al., 2001). Their ubiquitous occurrence, even in plants, suggests indispensible functions (Ohkura et al., 2001). XMAP215, for instance, is a promoter of microtubule elongation due to a suppression of catastrophe events induced by the Kin I family kinesin ...

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“…MT nucleation around chromosomes is mediated by a gradient of Ran-GTP that is highest near the chromosomes and diminishes away from the chromosomes (Carazo-Salas et al, 1999;Wilde and Zheng, 1999;Kalab et al, 2002Kalab et al, , 2006Caudron et al, 2005). Ran action in the spindle is mediated through nuclear localization signal (NLS)-containing spindle assembly factors that are kept in an inactive state by association with the nuclear import receptors importin ␣/␤ (Kalab and Heald, 2008;Walczak and Heald, 2008).…”

Section: Introductionmentioning

confidence: 99%

Kinesin-14 Family Proteins HSET/XCTK2 Control Spindle Length by Cross-Linking and Sliding Microtubules

Cai

Weaver

Ems-McClung

et al. 2009

MBoC

181

201

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Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin ␣/␤, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (

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Stimulation of Microtubule Aster Formation and Spindle Assembly by the Small GTPase Ran

Cited by 371 publications

References 23 publications

Mitosis-specific Anchoring of γ Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry

Mitosis-specific Anchoring of γ Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry

Regulated Expression of the Centrosomal Protein DdCP224 Affects Microtubule Dynamics and Reveals Mechanisms for the Control of Supernumerary Centrosome Number

Kinesin-14 Family Proteins HSET/XCTK2 Control Spindle Length by Cross-Linking and Sliding Microtubules

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