Aurora A site specific TACC3 phosphorylation regulates astral microtubule assembly by stabilizing γ-tubulin ring complex

Abstract Background Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. Results Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components. Conclusions The results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.

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Kinetochore microtubules shorten by loss of subunits at the kinetochores of prometaphase chromosomes

Journal of Cell Science ◽

10.1242/jcs.98.2.151 ◽

1991 ◽

Vol 98 (2) ◽

pp. 151-158 ◽

Cited By ~ 3

Author(s):

L. Cassimeris ◽

E.D. Salmon

Keyword(s):

Metaphase Plate ◽

Force Generation ◽

Microtubule Assembly ◽

Immunofluorescent Staining ◽

Chromosome Movement ◽

Mitotic Spindles ◽

Subunit Dissociation ◽

Spindle Poles ◽

Tubulin Subunit ◽

A Site

The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence photobleaching techniques and nocodazole-induced spindle shortening. Synchronous shortening of all kinetochore microtubules was produced by incubating cells in 17 microM nocodazole to block microtubule assembly. Under these conditions the spindle poles moved towards the metaphase plate at a rate of 3.6 +/− 0.4 microns min-1 (n = 3). On the basis of anti-tubulin immunofluorescent staining of cells fixed after incubation in nocodazole, we found that nonkinetochore microtubules rapidly disappeared and only kinetochore fibers were present after 60–90 s in nocodazole. To localize the site of tubulin subunit dissociation, a narrow bar pattern was photobleached across one half-spindle in prometaphase-metaphase cells previously microinjected with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF)-labeled tubulin. Immediately after photobleaching, cells were perfused with 17 microM nocodazole to produce shortening of kinetochore microtubules. Shortening was accompanied by a decrease in the distance between the bleach bar and the kinetochores. In contrast, there was little or no decrease in the distance between the bleach bar and the pole. Compared to their initial lengths, the average kinetochore to pole distance shortened by 18%, the bleach bar to kinetochore distance shortened by 28% and the average bleached bar to pole distance shortened by 1.6%. The data provide evidence that tubulin subunits dissociate from kinetochore microtubules at a site near the kinetochore during poleward chromosome movement. These results are consistent with models of poleward force generation for chromosome movement in which prometaphase-metaphase poleward force is generated in association with the kinetochore.

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Drosophila Aurora A kinase is required to localize D-TACC to centrosomes and to regulate astral microtubules

The Journal of Cell Biology ◽

10.1083/jcb.200108135 ◽

2002 ◽

Vol 156 (3) ◽

pp. 437-451 ◽

Cited By ~ 217

Author(s):

Régis Giet ◽

Doris McLean ◽

Simon Descamps ◽

Michael J. Lee ◽

Jordan W. Raff ◽

...

Keyword(s):

Coiled Coil ◽

Aurora Kinase ◽

The Other ◽

S2 Cells ◽

Aurora A Kinase ◽

Aurora A ◽

Spindle Poles

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.

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Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis

Journal of Cell Science ◽

10.1242/jcs.107.4.859 ◽

1994 ◽

Vol 107 (4) ◽

pp. 859-867 ◽

Cited By ~ 19

Author(s):

S.A. Endow ◽

R. Chandra ◽

D.J. Komma ◽

A.H. Yamamoto ◽

E.D. Salmon

Keyword(s):

Cell Cycle ◽

Coiled Coil ◽

Motor Protein ◽

Spindle Pole ◽

Opposite Polarity ◽

Microtubule Assembly ◽

Loss Of Function ◽

Spindle Poles ◽

Microtubule Motor ◽

Microtubule Motor Protein

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein required for meiotic and early mitotic chromosome distribution in Drosophila. ncd translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends, and is associated with spindles in chromosome/spindle preparations. Here we report a new mutant of ncd caused by partial deletion of the predicted coiled-coil central stalk. The mutant protein exhibits a velocity of translocation and ability to generate torque in motility assays comparable to near full-length ncd, but only partially rescues a null mutant for chromosome mis-segregation. Antibody staining experiments show that the partial loss-of-function and null mutants cause centrosomal and spindle pole defects, including centrosome splitting and loss of centrosomes from spindle poles, and localize ncd to centrosomes as well as spindles of wild-type embryos. Association of ncd with spindles and centrosomes is microtubule- and cell cycle-dependent: inhibition of microtubule assembly with colchicine abolishes ncd staining and centrosomal staining is observed in prometaphase, metaphase and anaphase, but diminishes in late anaphase/telophase. The cell cycle dependence of centrosomal staining and the defects of mutants provide clear evidence for activity of the ncd motor protein near or at the spindle poles in mitosis. The ncd motor may interact with centrosomal microtubules and spindle fibers to attach centrosomes to spindle poles, and mediate poleward translocation (flux) of kinetochore fibers, a process that may underlie poleward movement of chromosomes in mitosis. Together with previous work, our findings indicate that ncd is important in maintaining spindle poles in mitosis as well as in meiosis.

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Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring γ-Tubulin Ring Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0112 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3235-3245 ◽

Cited By ~ 160

Author(s):

Mikiko Takahashi ◽

Akiko Yamagiwa ◽

Tamako Nishimura ◽

Hideyuki Mukai ◽

Yoshitaka Ono

Keyword(s):

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Terminal Region ◽

Microtubule Assembly ◽

Microtubule Nucleation ◽

Amino Terminal ◽

Ring Complex ◽

Carboxyl Terminal

Microtubule assembly is initiated by the γ-tubulin ring complex (γ-TuRC). In yeast, the microtubule is nucleated from γ-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors γ-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with γ-tubulin through binding with γ-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and γ-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with γ-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring γ-TuRC.

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Recombination occurs within minutes of replication blockage by RTS1 producing restarted forks that are prone to collapse

eLife ◽

10.7554/elife.04539 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 29

Author(s):

Michael O Nguyen ◽

Manisha Jalan ◽

Carl A Morrow ◽

Fekret Osman ◽

Matthew C Whitby

Keyword(s):

Cell Cycle ◽

Replication Fork ◽

Genome Duplication ◽

Time Lapse ◽

Genetic Change ◽

Replication Proteins ◽

Site Specific ◽

Time Lapse Microscopy ◽

A Site ◽

Stalled Fork

The completion of genome duplication during the cell cycle is threatened by the presence of replication fork barriers (RFBs). Following collision with a RFB, replication proteins can dissociate from the stalled fork (fork collapse) rendering it incapable of further DNA synthesis unless recombination intervenes to restart replication. We use time-lapse microscopy and genetic assays to show that recombination is initiated within ∼10 min of replication fork blockage at a site-specific barrier in fission yeast, leading to a restarted fork within ∼60 min, which is only prevented/curtailed by the arrival of the opposing replication fork. The restarted fork is susceptible to further collapse causing hyper-recombination downstream of the barrier. Surprisingly, in our system fork restart is unnecessary for maintaining cell viability. Seemingly, the risk of failing to complete replication prior to mitosis is sufficient to warrant the induction of recombination even though it can cause deleterious genetic change.

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The Soundlapse Project: Exploring Spatiotemporal Features of Wetland Soundscapes

Leonardo ◽

10.1162/leon_a_02142 ◽

2021 ◽

pp. 1-9

Author(s):

Felipe Otondo ◽

André Rabello-Mestre

Keyword(s):

Time Lapse ◽

Spatial Audio ◽

Site Specific ◽

Sound Installation ◽

Field Recordings ◽

Future Developments ◽

Interdisciplinary Project ◽

Temporal Features ◽

Different Types ◽

A Site

Abstract The article discusses an interdisciplinary project aimed at highlighting the acoustical heritage of wetlands, by means of field recordings and a novel time-lapse montage method. We discuss a site-specific sound installation that was designed using original wetlands field recordings, live processing, and spatial audio multi-channel reproduction. The discussion focuses on spatial and temporal features of different types of recorded wetlands soundscapes. Future developments of this project will consider the implementation of a standalone spatiotemporal application, to be used in the context of virtual reality applications, game audio, and interactive dance performance.

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ST6Gal1 targets the ectodomain of ErbB2 in a site-specific manner and regulates gastric cancer cell sensitivity to trastuzumab

Oncogene ◽

10.1038/s41388-021-01801-w ◽

2021 ◽

Author(s):

Henrique O. Duarte ◽

Joana G. Rodrigues ◽

Catarina Gomes ◽

Paul J. Hensbergen ◽

Agnes L. Hipgrave Ederveen ◽

...

Keyword(s):

Gastric Cancer ◽

Molecular Mechanisms ◽

Clinical Performance ◽

Cell Surface Receptor ◽

Trastuzumab Resistance ◽

Site Specific ◽

Target Epitope ◽

Surface Receptor ◽

Unresectable Gastric Cancer ◽

A Site

AbstractThe clinical performance of the therapeutic monoclonal antibody trastuzumab in the treatment of ErbB2-positive unresectable gastric cancer (GC) is severely hampered by the emergence of molecular resistance. Trastuzumab’s target epitope is localized within the extracellular domain of the oncogenic cell surface receptor tyrosine kinase (RTK) ErbB2, which is known to undergo extensive N-linked glycosylation. However, the site-specific glycan repertoire of ErbB2, as well as the detailed molecular mechanisms through which specific aberrant glycan signatures functionally impact the malignant features of ErbB2-addicted GC cells, including the acquisition of trastuzumab resistance, remain elusive. Here, we demonstrate that ErbB2 is modified with both α2,6- and α2,3-sialylated glycan structures in GC clinical specimens. In-depth mass spectrometry-based glycomic and glycoproteomic analysis of ErbB2’s ectodomain disclosed a site-specific glycosylation profile in GC cells, in which the ST6Gal1 sialyltransferase specifically targets ErbB2 N-glycosylation sites occurring within the receptor’s trastuzumab-binding domain. Abrogation of ST6Gal1 expression reshaped the cellular and ErbB2-specific glycomes, expanded the cellular half-life of the ErbB2 receptor, and sensitized ErbB2-dependent GC cells to trastuzumab-induced cytotoxicity through the stabilization of ErbB dimers at the cell membrane, and the decreased activation of both ErbB2 and EGFR RTKs. Overall, our data demonstrates that ST6Gal1-mediated aberrant α2,6-sialylation actively tunes the resistance of ErbB2-driven GC cells to trastuzumab.

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