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 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 A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression.

1990 ◽  
Vol 111 (2) ◽  
pp. 511-522 ◽  
Author(s):  
C Nislow ◽  
C Sellitto ◽  
R Kuriyama ◽  
J R McIntosh
Keyword(s):  
Monoclonal Antibody ◽  
Protein S ◽  
Dependent Manner ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Microtubule Organization ◽  
Cell Extracts ◽  
Mitotic Inhibition ◽  
Specific Localization ◽  
Dose Dependent

A monoclonal antibody raised against mitotic spindles isolated from CHO cells ([CHO1], Sellitto, C., and R. Kuriyama. 1988. J. Cell Biol. 106:431-439) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Parallel control injections with nonimmune IgMs do not yield significant mitotic inhibition. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase.

scholarly journals Active elimination of intestinal cells drives oncogenic growth in organoids

2020 ◽  
Author(s):  
Ana Krotenberg Garcia ◽  
Arianna Fumagalli ◽  
Huy Quang Le ◽  
Owen J. Sansom ◽  
Jacco van Rheenen ◽  
...  
Keyword(s):  
Quality Control ◽  
Cell Death ◽  
Cancer Cells ◽  
Cell Fate ◽  
Crucial Role ◽  
Intestinal Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Cell Competition ◽  
Type Population

AbstractCompetitive cell-interactions play a crucial role in quality control during development and homeostasis. Here we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell fate transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. JNK signalling is activated in competing cells and is required for cell fate change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell fate change in a JNK dependent manner.

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 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 Decorin induced by progesterone plays a crucial role in suppressing endometriosis

2014 ◽  
Vol 223 (2) ◽  
pp. 203-216 ◽  
Author(s):  
Yoshihiro Joshua Ono ◽  
Yoshito Terai ◽  
Akiko Tanabe ◽  
Atsushi Hayashi ◽  
Masami Hayashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mrna Expression ◽  
Cell Lines ◽  
Stromal Cells ◽  
Crucial Role ◽  
Dependent Manner ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Cycle Arrest ◽  
Dose Dependent

Dienogest, a synthetic progestin, has been shown to be effective against endometriosis, although it is still unclear as to how it affects the ectopic endometrial cells. Decorin has been shown to be a powerful endogenous tumor repressor acting in a paracrine fashion to limit tumor growth. Our objectives were to examine the direct effects of progesterone and dienogest on the in vitro proliferation of the human ectopic endometrial epithelial and stromal cell lines, and evaluate as to how decorin contributes to this effect. We also examined DCN mRNA expression in 50 endometriosis patients. The growth of both cell lines was inhibited in a dose-dependent manner by both decorin and dienogest. Using a chromatin immunoprecipitation assay, it was noted that progesterone and dienogest directly induced the binding of the decorin promoter in the EMOsis cc/TERT cells (immortalized human ovarian epithelial cells) and CRL-4003 cells (immortalized human endometrial stromal cells). Progesterone and dienogest also led to significant induced cell cycle arrest via decorin by promoting production of p21 in both cell lines in a dose-dependent manner. Decorin also suppressed the expression of MET in both cell lines. We confirmed that DCN mRNA expression in patients treated with dienogest was higher than that in the control group. In conclusion, decorin induced by dienogest appears to play a crucial role in suppressing endometriosis by exerting anti-proliferative effects and inducing cell cycle arrest via the production of p21 human ectopic endometrial cells and eutopic endometrial stromal cells.

scholarly journals Distinct Dgrip84 Isoforms Correlate with Distinct γ-Tubulins in Drosophila

2008 ◽  
Vol 19 (1) ◽  
pp. 368-377 ◽  
Author(s):  
Christiane Wiese
Keyword(s):  
Cell Cycle ◽  
Spindle Pole ◽  
Multiprotein Complex ◽  
Microtubule Organization ◽  
Developmentally Regulated ◽  
Tubulin Genes ◽  
Tubulin Isotype ◽  
Ring Complex ◽  
Cycle Dependent ◽  
Drosophila Embryos

γ-Tubulin is an indispensable component of the animal centrosome and is required for proper microtubule organization. Within the cell, γ-tubulin exists in a multiprotein complex containing between two (some yeasts) and six or more (metazoa) additional highly conserved proteins named gamma ring proteins (Grips) or gamma complex proteins (GCPs). γ-Tubulin containing complexes isolated from Xenopus eggs or Drosophila embryos appear ring-shaped and have therefore been named the γ-tubulin ring complex (γTuRC). Curiously, many organisms (including humans) have two distinct γ-tubulin genes. In Drosophila, where the two γ-tubulin isotypes have been studied most extensively, the γ-tubulin genes are developmentally regulated: the “maternal” γ-tubulin isotype (named γTub37CD according to its location on the genetic map) is expressed in the ovary and is deposited in the egg, where it is thought to orchestrate the meiotic and early embryonic cleavages. The second γ-tubulin isotype (γTub23C) is ubiquitously expressed and persists in most of the cells of the adult fly. In those rare cases where both γ-tubulins coexist in the same cell, they show distinct subcellular distributions and cell-cycle-dependent changes: γTub37CD mainly localizes to the centrosome, where its levels vary only slightly with the cell cycle. In contrast, the level of γTub23C at the centrosome increases at the beginning of mitosis, and γTub23C also associates with spindle pole microtubules. Here, we show that γTub23C forms discrete complexes that closely resemble the complexes formed by γTub37CD. Surprisingly, however, γTub23C associates with a distinct, longer splice variant of Dgrip84. This may reflect a role for Dgrip84 in regulating the activity and/or the location of the γ-tubulin complexes formed with γTub37CD and γTub23C.

A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase

2009 ◽  
Vol 419 (2) ◽  
pp. 329-338 ◽  
Author(s):  
Masahiko Taura ◽  
Kei Miyano ◽  
Reiko Minakami ◽  
Sachiko Kamakura ◽  
Ryu Takeya ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Crucial Role ◽  
Sh3 Domain ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Wild Type ◽  
Catalytic Core ◽  
Membrane Recruitment ◽  
Phagocyte Nadph Oxidase ◽  
Isoleucine Residue

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox–p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

scholarly journals The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis.

1986 ◽  
Vol 102 (5) ◽  
pp. 1688-1698 ◽  
Author(s):  
L Wordeman ◽  
K L McDonald ◽  
W Z Cande
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Centric Diatom ◽  
Mitotic Spindles ◽  
Animal Cells ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Microtubule ◽  
Cytoplasmic Microtubules

The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.

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