scholarly journals Aurora A contributes to p150glued phosphorylation and function during mitosis

2010 ◽  
Vol 189 (4) ◽  
pp. 651-659 ◽  
Author(s):  
Pierre Romé ◽  
Emilie Montembault ◽  
Nathalie Franck ◽  
Aude Pascal ◽  
David M. Glover ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Microtubule Binding ◽  
Microtubule Binding Domain ◽  
Spindle Microtubules ◽  
And Function

Aurora A is a spindle pole–associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit p150glued on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A's function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the p150glued. Although wild-type p150glued binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous p150glued. Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the p150glued MBD to limit the microtubule binding of the dynein–dynactin complex and thus regulates spindle assembly.

Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin

2000 ◽  
Vol 113 (9) ◽  
pp. 1623-1633 ◽  
Author(s):  
K.P. McNally ◽  
O.A. Bazirgan ◽  
F.J. McNally
Keyword(s):  
Mitotic Spindle ◽  
Binding Proteins ◽  
Spindle Pole ◽  
Negative Regulator ◽  
Microtubule Binding ◽  
Microtubule Severing ◽  
Spindle Poles ◽  
Wd40 Domain

The assembly and function of the mitotic spindle requires the activity of a number of microtubule-binding proteins. Some microtubule-binding proteins bind microtubules in vitro but do not co-localize with microtubules in interphase cells. Instead these proteins associate with specific subregions of the mitotic spindle. Katanin, a heterodimeric microtubule-severing ATPase, is found localized at mitotic spindle poles. In this paper we demonstrate that human p60 katanin and the C-terminal domain of human p80 katanin both bind microtubules in vitro. Association of these two proteins results in an increased microtubule affinity and increased microtubule-severing activity in vitro. Association of these subunits in transfected HeLa cells increases microtubule disassembly activity and targeting to spindle poles. The N-terminal WD40 domain of p80 katanin acts as a negative regulator of microtubule disassembly activity and is also required for spindle pole localization, possibly through interactions with another spindle-pole protein. These results support a model in which katanin is targeted to spindle poles through a combination of direct microtubule binding by the p60 subunit and through interactions between the WD40 domain and an unknown protein. We propose that both domains of p80 are essential in precisely regulating katanin's activity in vivo.

Katanin, the microtubule-severing ATPase, is concentrated at centrosomes

1996 ◽  
Vol 109 (3) ◽  
pp. 561-567 ◽  
Author(s):  
F.J. McNally ◽  
K. Okawa ◽  
A. Iwamatsu ◽  
R.D. Vale
Keyword(s):  
Mitotic Spindle ◽  
Dynamic Properties ◽  
Mitotic Spindles ◽  
Microtubule Severing ◽  
Spindle Microtubules ◽  
Gamma Tubulin ◽  
And Function ◽  
Poleward Flux

The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.

scholarly journals Aurora at the pole and equator: overlapping functions of Aurora kinases in the mitotic spindle

Open Biology ◽  
2013 ◽  
Vol 3 (3) ◽  
pp. 120185 ◽  
Author(s):  
Helfrid Hochegger ◽  
Nadia Hégarat ◽  
Jose B. Pereira-Leal
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Aurora Kinase ◽  
Spindle Pole ◽  
Aurora A ◽  
Mitotic Progression ◽  
Aurora Kinases ◽  
Mitotic Kinases ◽  
Chromosome Alignment ◽  
Spindle Microtubules

The correct assembly and timely disassembly of the mitotic spindle is crucial for the propagation of the genome during cell division. Aurora kinases play a central role in orchestrating bipolar spindle establishment, chromosome alignment and segregation. In most eukaryotes, ranging from amoebas to humans, Aurora activity appears to be required both at the spindle pole and the kinetochore, and these activities are often split between two different Aurora paralogues, termed Aurora A and B. Polar and equatorial functions of Aurora kinases have generally been considered separately, with Aurora A being mostly involved in centrosome dynamics, whereas Aurora B coordinates kinetochore attachment and cytokinesis. However, double inactivation of both Aurora A and B results in a dramatic synergy that abolishes chromosome segregation. This suggests that these two activities jointly coordinate mitotic progression. Accordingly, recent evidence suggests that Aurora A and B work together in both spindle assembly in metaphase and disassembly in anaphase. Here, we provide an outlook on these shared functions of the Auroras, discuss the evolution of this family of mitotic kinases and speculate why Aurora kinase activity may be required at both ends of the spindle microtubules.

scholarly journals Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 374 ◽  
Author(s):  
Francesco D. Naso ◽  
Valentina Sterbini ◽  
Elena Crecca ◽  
Italia A. Asteriti ◽  
Alessandra D. Russo ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Transformed Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Truncated Form ◽  
Daughter Cells ◽  
Lamin B1 ◽  
Mitotic Spindle Assembly ◽  
Nuclear Reconstitution

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent—or independent—on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.

scholarly journals The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

1999 ◽  
Vol 147 (2) ◽  
pp. 351-366 ◽  
Author(s):  
Vicki Mountain ◽  
Calvin Simerly ◽  
Louisa Howard ◽  
Asako Ando ◽  
Gerald Schatten ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cultured Cells ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Cross Linking ◽  
Simultaneous Inhibition ◽  
Balance Of Forces ◽  
And Function

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

scholarly journals Aurora A Regulates the Activity of HURP by Controlling the Accessibility of Its Microtubule-binding Domain

2008 ◽  
Vol 19 (5) ◽  
pp. 2083-2091 ◽  
Author(s):  
Jim Wong ◽  
Robert Lerrigo ◽  
Chang-Young Jang ◽  
Guowei Fang
Keyword(s):  
Ectopic Expression ◽  
Binding Activity ◽  
Binding Domain ◽  
Aurora A ◽  
Microtubule Binding ◽  
Physiological Importance ◽  
Mitotic Kinase ◽  
Terminal Domain ◽  
Microtubule Binding Domain ◽  
And Function

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

scholarly journals Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Kimberly K. Fong ◽  
Trisha N. Davis ◽  
Charles L. Asbury
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Force Generation ◽  
Initial Contact ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules ◽  
Pole Component

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.

scholarly journals Saccharomyces cerevisiae Duo1p and Dam1p, Novel Proteins Involved in Mitotic Spindle Function

1998 ◽  
Vol 143 (4) ◽  
pp. 1029-1040 ◽  
Author(s):  
Christian Hofmann ◽  
Iain M. Cheeseman ◽  
Bruce L. Goode ◽  
Kent L. McDonald ◽  
Georjana Barnes ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Fluorescent Protein ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Spindle Pole Bodies ◽  
Intranuclear Microtubules ◽  
Spindle Microtubules ◽  
Green Fluorescent

In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP–Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.

scholarly journals A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma

Blood ◽  
2010 ◽  
Vol 115 (25) ◽  
pp. 5202-5213 ◽  
Author(s):  
Güllü Görgün ◽  
Elisabetta Calabrese ◽  
Teru Hideshima ◽  
Jeffrey Ecsedy ◽  
Giulia Perrone ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mitotic Spindle ◽  
Kinase Inhibitor ◽  
Phase 1 ◽  
Tumor Burden ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Kinase

Abstract Aurora-A is a mitotic kinase that regulates mitotic spindle formation and segregation. In multiple myeloma (MM), high Aurora-A gene expression has been correlated with centrosome amplification and proliferation; thus, inhibition of Aurora-A in MM may prove to be therapeutically beneficial. Here we assess the in vitro and in vivo anti-MM activity of MLN8237, a small-molecule Aurora-A kinase inhibitor. Treatment of cultured MM cells with MLN8237 results in mitotic spindle abnormalities, mitotic accumulation, as well as inhibition of cell proliferation through apoptosis and senescence. In addition, MLN8237 up-regulates p53 and tumor suppressor genes p21 and p27. Combining MLN8237 with dexamethasone, doxorubicin, or bortezomib induces synergistic/additive anti-MM activity in vitro. In vivo anti-MM activity of MLN8237 was confirmed using a xenograft-murine model of human-MM. Tumor burden was significantly reduced (P = .007) and overall survival was significantly increased (P < .005) in animals treated with 30 mg/kg MLN8237 for 21 days. Induction of apoptosis and cell death by MLN8237 were confirmed in tumor cells excised from treated animals by TdT-mediated dUTP nick end labeling assay. MLN8237 is currently in phase 1 and phase 2 clinical trials in patients with advanced malignancies, and our preclinical results suggest that MLN8237 may be a promising novel targeted therapy in MM.

scholarly journals The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation

2015 ◽  
Vol 26 (19) ◽  
pp. 3424-3438 ◽  
Author(s):  
Sara Carvalhal ◽  
Susana Abreu Ribeiro ◽  
Miguel Arocena ◽  
Taciana Kasciukovic ◽  
Achim Temme ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Nuclear Pore ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Triple A Syndrome ◽  
Mitotic Kinases ◽  
Nuclear Pore Protein ◽  
Complex Process ◽  
Chromosome Alignment

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome.

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