scholarly journals CDK5RAP2 stimulates microtubule nucleation by the γ-tubulin ring complex

2010 ◽  
Vol 191 (6) ◽  
pp. 1089-1095 ◽  
Author(s):  
Yuk-Kwan Choi ◽  
Pengfei Liu ◽  
Siu Kwan Sze ◽  
Chao Dai ◽  
Robert Z. Qi
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Schizosaccharomyces Pombe ◽  
Binding Domain ◽  
Deficient Mutant ◽  
Microtubule Nucleation ◽  
Ring Complex ◽  
Regulatory Functions

CDK5RAP2 is a human microcephaly protein that contains a γ-tubulin complex (γ-TuC)–binding domain conserved in Drosophila melanogaster centrosomin and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC–tethering proteins. In this study, we show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC–mediated nucleation activator (γ-TuNA). γ-TuNA but not its γ-TuC–binding-deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2–6. RNA interference–mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC–mediated microtubule nucleation.

scholarly journals Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

1998 ◽  
Vol 142 (3) ◽  
pp. 775-786 ◽  
Author(s):  
Michelle Moritz ◽  
Yixian Zheng ◽  
Bruce M. Alberts ◽  
Karen Oegema
Keyword(s):  
Soluble Fraction ◽  
Protein Complexes ◽  
Drosophila Embryo ◽  
Microtubule Nucleation ◽  
Complementation Assay ◽  
Embryo Extract ◽  
Ring Complex ◽  
Centrosomal Proteins

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.

scholarly journals Characterization of a Drosophila Centrosome Protein CP309 That Shares Homology with Kendrin and CG-NAP

2004 ◽  
Vol 15 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Shin-ichi Kawaguchi ◽  
Yixian Zheng
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Biochemical Studies ◽  
Ring Complex ◽  
Centrosome Protein

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the γ-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the γ-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.

scholarly journals γ-Tubulin ring complexes regulate microtubule plus end dynamics

2009 ◽  
Vol 187 (3) ◽  
pp. 327-334 ◽  
Author(s):  
Anaïs Bouissou ◽  
Christel Vérollet ◽  
Aureliana Sousa ◽  
Paula Sampaio ◽  
Michel Wright ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Live Imaging ◽  
S2 Cells ◽  
Imaging Analysis ◽  
Mitotic Progression ◽  
Small Complex ◽  
Drosophila S2 Cells ◽  
Ring Complex ◽  
Ring Complexes

γ-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the γ-tubulin small complex (γ-TuSC) and the γ-tubulin ring complex (γ-TuRC). Proteins specific of the γ-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during interphase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the γ-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the γ-TuRC localizes along interphase MTs and that distal γ-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the γ-TuRC associated to MTs may regulate their dynamics by limiting catastrophes.

scholarly journals MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template

2016 ◽  
Vol 215 (6) ◽  
pp. 823-840 ◽  
Author(s):  
Tien-chen Lin ◽  
Annett Neuner ◽  
Dirk Flemming ◽  
Peng Liu ◽  
Takumi Chinen ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Human Cells ◽  
Molecular Function ◽  
Microtubule Nucleation ◽  
Small Complex ◽  
Ring Complex

MOZART1/Mzt1 is required for the localization of γ-tubulin complexes to microtubule (MT)–organizing centers from yeast to human cells. Nevertheless, the molecular function of MOZART1/Mzt1 is largely unknown. Taking advantage of the minimal MT nucleation system of Candida albicans, we reconstituted the interactions of Mzt1, γ-tubulin small complex (γ-TuSC), and γ-tubulin complex receptors (γ-TuCRs) Spc72 and Spc110 in vitro. With affinity measurements, domain deletion, and swapping, we show that Spc110 and Mzt1 bind to distinct regions of the γ-TuSC. In contrast, both Mzt1 and γ-TuSC interact with the conserved CM1 motif of Spc110/Spc72. Spc110/Spc72 and Mzt1 constitute “oligomerization chaperones,” cooperatively promoting and directing γ-TuSC oligomerization into MT nucleation-competent rings. Consistent with the functions of Mzt1, human MOZART1 directly interacts with the CM1-containing region of the γ-TuCR CEP215. MOZART1 depletion in human cells destabilizes the large γ-tubulin ring complex and abolishes CEP215CM1-induced ectopic MT nucleation. Together, we reveal conserved functions of MOZART1/Mzt1 through interactions with γ-tubulin complex subunits and γ-TuCRs.

scholarly journals An in Vivo Analysis of the vestigial Gene in Drosophila melanogaster Defines the Domains Required for Vg Function

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1365-1373 ◽  
Author(s):  
Julie O MacKay ◽  
Kelly H Soanes ◽  
Ajay Srivastava ◽  
Andrew Simmonds ◽  
William J Brook ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Domain ◽  
Wing Development ◽  
Amino Acid Motif ◽  
Considerable Evidence ◽  
Cellular Context ◽  
In Vivo Analysis ◽  
Necessary And Sufficient

Abstract Considerable evidence indicates an obligate partnership of the Drosophila melanogaster Vestigial (VG) and Scalloped (SD) proteins within the context of wing development. These two proteins interact physically and a 56-amino-acid motif within VG is necessary and sufficient for this binding. While the importance of this SD-binding domain has been clearly demonstrated both in vitro and in vivo, the remaining portions of VG have not been examined for in vivo function. Herein, additional regions within VG were tested for possible in vivo functions. The results identify two additional domains that must be present for optimal VG function as measured by the loss of ability to rescue vg mutants, to induce ectopic sd expression, and to perform other normal VG functions when they are deleted. An in vivo study such as this one is fundamentally important because it identifies domains of VG that are necessary in the cellular context in which wing development actually occurs. The results also indicate that an additional large portion of VG, outside of these two domains and the SD-binding domain, is dispensable in the execution of these normal VG functions.

scholarly journals Structural analysis of the role of TPX2 in branching microtubule nucleation

2017 ◽  
Vol 216 (4) ◽  
pp. 983-997 ◽  
Author(s):  
Raymundo Alfaro-Aco ◽  
Akanksha Thawani ◽  
Sabine Petry
Keyword(s):  
Structural Analysis ◽  
Xenopus Laevis ◽  
Mitotic Spindle ◽  
Microtubule Nucleation ◽  
Domain Organization ◽  
Ring Complex ◽  
The Right

The mitotic spindle consists of microtubules (MTs), which are nucleated by the γ-tubulin ring complex (γ-TuRC). How the γ-TuRC gets activated at the right time and location remains elusive. Recently, it was uncovered that MTs nucleate from preexisting MTs within the mitotic spindle, which requires the protein TPX2, but the mechanism basis for TPX2 action is unknown. Here, we investigate the role of TPX2 in branching MT nucleation. We establish the domain organization of Xenopus laevis TPX2 and define the minimal TPX2 version that stimulates branching MT nucleation, which we find is unrelated to TPX2’s ability to nucleate MTs in vitro. Several domains of TPX2 contribute to its MT-binding and bundling activities. However, the property necessary for TPX2 to induce branching MT nucleation is contained within newly identified γ-TuRC nucleation activator motifs. Separation-of-function mutations leave the binding of TPX2 to γ-TuRC intact, whereas branching MT nucleation is abolished, suggesting that TPX2 may activate γ-TuRC to promote branching MT nucleation.

RNA interference and its clinical applications

2007 ◽  
Vol 148 (47) ◽  
pp. 2235-2240 ◽  
Author(s):  
Gyöngyi Munkácsy ◽  
Zsolt Tulassay ◽  
Balázs Győrffy
Keyword(s):  
Rna Interference ◽  
Clinical Applications

Az RNS-interferencia a poszttranszkripciós génelcsendesítés olyan formája, amelynek során rövid, specifikusan RNS-molekulák elnyomják a gének kifejeződésében kulcsszerepet játszó hírvivő RNS-ek működését. A sejtbe juttatott dupla szálú vagy rövid interferáló RNS-molekulák aktiválják az RNS-indukált elcsendesítő komplexet, amely a célgén hírvivő RNS-ét lebontja. A sejtek saját szabályozó mikro-RNS-molekulákkal is rendelkeznek, amelyeknek hírvivő RNS-e képes önmagával hajtűt képezni, amit a sejt dupla szálú RNS-ként értelmez. Az RNS-interferencia élettani működései közé tartozik a vírusok és a transzpozonok elleni védekezés, valamint a génkifejeződés szabályozása. Az RNS-interferencia nemcsak in vitro alkalmazható az egyes gének működésének vizsgálatára, hanem klinikai alkalmazásainak lehetőségei is megjelentek. Eddig vírusfertőzésekben, az időskori makuladegeneráció gátlására, a vér koleszterinszint-csökkentésére, daganatellenes és neurodegeneratív betegségek kezelésében alkalmazták. Az RNS-interferencia alkalmazását azonban nehezíti, hogy a megfelelő rövid interferáló RNS-molekulák tervezéséhez szükséges bioinformatikai algoritmusok nem tökéletesek; a szervezet szöveteibe való bejuttatásuk nehéz; illetve csak olyan esetekben alkalmazható, amelyekben átmeneti antagonista génelcsendesítő hatás és nem hosszú távú kezelés szükséges. Az alkalmazás legnagyobb előnye a jelentős specificitás, ami miatt mellékhatása is kevés. Az RNS-interferencia alapú kezelések megjelenése már a közeli jövőben várható.

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