scholarly journals NEDD1-dependent recruitment of the γ-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly

2006 ◽  
Vol 172 (4) ◽  
pp. 505-515 ◽  
Author(s):  
Laurence Haren ◽  
Marie-Hélène Remy ◽  
Ingrid Bazin ◽  
Isabelle Callebaut ◽  
Michel Wright ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Somatic Cells ◽  
Spindle Assembly ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Centrosomal Protein ◽  
Centriole Duplication ◽  
Large Complex ◽  
Ring Complex ◽  
Carboxy Terminal

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein γ-tubulin. In mammals, γ-tubulin associates with additional proteins into a large complex, the γ-tubulin ring complex (γTuRC). We characterize NEDD1, a centrosomal protein that associates with γTuRCs. We show that the majority of γTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of γ-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to γTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of γ-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of γ-tubulin to the centrosome.

scholarly journals The centriolar satellite protein SSX2IP promotes centrosome maturation

2013 ◽  
Vol 202 (1) ◽  
pp. 81-95 ◽  
Author(s):  
Felix Bärenz ◽  
Daigo Inoue ◽  
Hideki Yokoyama ◽  
Justus Tegha-Dunghu ◽  
Stephanie Freiss ◽  
...  
Keyword(s):  
Somatic Cells ◽  
Spindle Assembly ◽  
Xenopus Laevis Oocyte ◽  
Dependent Manner ◽  
Vertebrate Development ◽  
Spindle Poles ◽  
M Phase ◽  
Egg Extracts ◽  
Ring Complex ◽  
Pericentriolar Material

Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.

scholarly journals Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

2003 ◽  
Vol 162 (5) ◽  
pp. 757-764 ◽  
Author(s):  
Yasuhiko Terada ◽  
Yumi Uetake ◽  
Ryoko Kuriyama
Keyword(s):  
Mammalian Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Spindle Poles

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

scholarly journals CDK5RAP2 Is a Pericentriolar Protein That Functions in Centrosomal Attachment of the γ-Tubulin Ring Complex

2008 ◽  
Vol 19 (1) ◽  
pp. 115-125 ◽  
Author(s):  
Ka-Wing Fong ◽  
Yuk-Kwan Choi ◽  
Jerome B. Rattner ◽  
Robert Z. Qi
Keyword(s):  
Autosomal Recessive ◽  
Macromolecular Complex ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Primary Microcephaly ◽  
Conserved Sequence ◽  
Ring Complex ◽  
Pericentriolar Material ◽  
Related Proteins ◽  
Autosomal Recessive Primary Microcephaly

Microtubule nucleation and organization by the centrosome require γ-tubulin, a protein that exists in a macromolecular complex called the γ-tubulin ring complex (γTuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including γ-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the γTuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for γTuRC attachment to the centrosome but not for γTuRC assembly. Perturbing CDK5RAP2 function delocalized γ-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in γTuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly.

scholarly journals Biochemical reconstitution of branching microtubule nucleation

2019 ◽  
Author(s):  
Raymundo Alfaro-Aco ◽  
Akanksha Thawani ◽  
Sabine Petry
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Chromosome Segregation ◽  
Mode Of Action ◽  
Spindle Assembly ◽  
Nucleation Site ◽  
Microtubule Nucleation ◽  
Ring Complex ◽  
Gamma Tubulin

AbstractMicrotubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways still need to be identified along with their mode of action. Here, using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, a nucleation pathway where microtubules originate from pre-existing microtubules, which is essential for spindle assembly and chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the two branching effectors augmin and TPX2 are required to efficiently nucleate branched microtubules. Specifically, TPX2 generates regularly-spaced patches that recruit augmin and γ-TuRC to microtubules, which then nucleate new microtubules at preferred branching angles of less than 90 degrees. Our work demonstrates how γ-TuRC is brought to its nucleation site for branching microtubule nucleation. It provides a blueprint for other microtubule nucleation pathways and for generating a particular microtubule architecture by regulating microtubule nucleation.

scholarly journals NME7 is a functional component of the γ-tubulin ring complex

2014 ◽  
Vol 25 (13) ◽  
pp. 2017-2025 ◽  
Author(s):  
Pengfei Liu ◽  
Yuk-Kwan Choi ◽  
Robert Z. Qi
Keyword(s):  
Cell Cycle ◽  
Crucial Role ◽  
Dependent Manner ◽  
Mitotic Spindles ◽  
Wild Type ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Functional Component ◽  
Ring Complex

As the primary microtubule nucleator in animal cells, the γ-tubulin ring complex (γTuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the γTuRC is regulated. Recently, isolated γTuRC was found to contain NME7, a poorly characterized member of the NME family. Here we report that NME7 is a γTuRC component that regulates the microtubule-nucleating activity of the γTuRC. NME7 contains two putative kinase domains, A and B, and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the γTuRC through both A and B domains, with Arg-322 in domain B being crucial to the binding. In association with the γTuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect γTuRC assembly or localization to centrosomes, but it does impair centrosome-based microtubule nucleation. Of importance, wild-type NME7 promotes γTuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the γTuRC in a kinase-dependent manner to facilitate microtubule nucleation.

scholarly journals Ccdc61 controls centrosomal localization of Cep170 and is required for spindle assembly and symmetry

2018 ◽  
Vol 29 (26) ◽  
pp. 3105-3118 ◽  
Author(s):  
Felix Bärenz ◽  
Yvonne T. Kschonsak ◽  
Annalena Meyer ◽  
Aliakbar Jafarpour ◽  
Holger Lorenz ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Microtubule Stability ◽  
Coiled Coil Domain ◽  
Intrinsic Symmetry ◽  
Spindle Microtubules

Microtubule nucleation was uncovered as a key principle of spindle assembly. However, the mechanistic details about microtubule nucleation and the organization of spindle formation and symmetry are currently being revealed. Here we describe the function of coiled-coil domain containing 61 (Ccdc61), a so far uncharacterized centrosomal protein, in spindle assembly and symmetry. Our data describe that Ccdc61 is required for spindle assembly and precise chromosome alignments in mitosis. Microtubule tip-tracking experiments in the absence of Ccdc61 reveal a clear loss of the intrinsic symmetry of microtubule tracks within the spindle. Furthermore, we show that Ccdc61 controls the centrosomal localization of centrosomal protein of 170 kDa (Cep170), a protein that was shown previously to localize to centrosomes as well as spindle microtubules and promotes microtubule organization and microtubule assembly. Interestingly, selective disruption of Ccdc61 impairs the binding between Cep170 and TANK binding kinase 1, an interaction that is required for microtubule stability. In summary, we have discovered Ccdc61 as a centrosomal protein with an important function in mitotic microtubule organization.

scholarly journals Microtubule assembly in meiotic extract requires glycogen

2011 ◽  
Vol 22 (17) ◽  
pp. 3139-3151 ◽  
Author(s):  
Aaron C. Groen ◽  
Margaret Coughlin ◽  
Timothy J. Mitchison
Keyword(s):  
Protein Complexes ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Microtubule Nucleation ◽  
Bipolar Spindle ◽  
Egg Extracts ◽  
Ring Complex

The assembly of microtubules during mitosis requires many identified components, such as γ-tubulin ring complex (γ-TuRC), components of the Ran pathway (e.g., TPX2, HuRP, and Rae1), and XMAP215/chTOG. However, it is far from clear how these factors function together or whether more factors exist. In this study, we used biochemistry to attempt to identify active microtubule nucleation protein complexes from Xenopus meiotic egg extracts. Unexpectedly, we found both microtubule assembly and bipolar spindle assembly required glycogen, which acted both as a crowding agent and as metabolic source glucose. By also reconstituting microtubule assembly in clarified extracts, we showed microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly.

scholarly journals Characterization of the TPX2 Domains Involved in Microtubule Nucleation and Spindle Assembly in Xenopus Egg Extracts

2004 ◽  
Vol 15 (12) ◽  
pp. 5318-5328 ◽  
Author(s):  
Stéphane Brunet ◽  
Teresa Sardon ◽  
Timo Zimmerman ◽  
Torsten Wittmann ◽  
Rainer Pepperkok ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Microtubule Nucleation ◽  
Terminal Domain ◽  
Large N ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Domain Functional ◽  
Xenopus Egg Extracts

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.

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