scholarly journals The Cep192-Organized Aurora A-Plk1 Cascade Is Essential for Centrosome Cycle and Bipolar Spindle Assembly

2014 ◽  
Vol 55 (4) ◽  
pp. 578-591 ◽  
Author(s):  
Vladimir Joukov ◽  
Johannes C. Walter ◽  
Arcangela De Nicolo
Keyword(s):  
Spindle Assembly ◽  
Aurora A ◽  
Bipolar Spindle

scholarly journals Kinetochore-generated pushing forces separate centrosomes during bipolar spindle assembly

2009 ◽  
Vol 184 (3) ◽  
pp. 365-372 ◽  
Author(s):  
Alberto Toso ◽  
Jennifer R. Winter ◽  
Ainslie J. Garrod ◽  
Ana C. Amaro ◽  
Patrick Meraldi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Envelope ◽  
Somatic Cells ◽  
Spindle Assembly ◽  
Aurora A ◽  
Bipolar Spindle ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
Centrosome Separation ◽  
Dependent Pathway

In animal somatic cells, bipolar spindle formation requires separation of the centrosome-based spindle poles. Centrosome separation relies on multiple pathways, including cortical forces and antiparallel microtubule (MT) sliding, which are two activities controlled by the protein kinase aurora A. We previously found that depletion of the human kinetochore protein Mcm21RCENP-O results in monopolar spindles, raising the question as to whether kinetochores contribute to centrosome separation. In this study, we demonstrate that kinetochores promote centrosome separation after nuclear envelope breakdown by exerting a pushing force on the kinetochore fibers (k-fibers), which are bundles of MTs that connect kinetochores to centrosomes. This force is based on poleward MT flux, which incorporates new tubulin subunits at the plus ends of k-fibers and requires stable k-fibers to drive centrosomes apart. This kinetochore-dependent force becomes essential for centrosome separation if aurora A is inhibited. We conclude that two mechanisms control centrosome separation during prometaphase: an aurora A–dependent pathway and a kinetochore-dependent pathway that relies on k-fiber–generated pushing forces.

scholarly journals The impact of chromosomes and centrosomes on spindle assembly as observed in living cells.

1995 ◽  
Vol 129 (5) ◽  
pp. 1287-1300 ◽  
Author(s):  
D Zhang ◽  
R B Nicklas
Keyword(s):  
Spindle Assembly ◽  
Living Cells ◽  
Microtubule Dynamics ◽  
Specific Site ◽  
Polarization Microscopy ◽  
Spindle Microtubule ◽  
Single Chromosome ◽  
Bipolar Spindle ◽  
The Impact

We analyzed the role that chromosomes, kinetochores, and centrosomes play in spindle assembly in living grasshopper spermatocytes by reconstructing spindles lacking certain components. We used video-enhanced, polarization microscopy to distinguish the effect of each component on spindle microtubule dynamics and we discovered that both chromosomes and centrosomes make potent and very different contributions to the organization of the spindle. Remarkably, the position of a single chromosome can markedly affect the distribution of microtubules within a spindle or even alter the fate of spindle assembly. In an experimentally constructed spindle having only one chromosome, moving the chromosome to one of the two poles induces a dramatic assembly of microtubules at the nearer pole and a concomitant disassembly at the farther pole. So long as a spindle carries a single chromosome it will persist normally. A spindle will also persist even when all chromosomes are detached and then removed from the cell. If, however, a single chromosome remains in the cell but is detached from the spindle and kept in the cytoplasm, the spindle disassembles. One might expect the effect of chromosomes on spindle assembly to relate to a property of a specific site on each chromosome, perhaps the kinetochore. We have ruled out that possibility by showing that it is the size of chromosomes rather than the number of kinetochores that matters. Although chromosomes affect spindle assembly, they cannot organize a spindle in the absence of centrosomes. In contrast, centrosomes can organize a functional bipolar spindle in the absence of chromosomes. If both centrosomes and chromosomes are removed from the cell, the spindle quickly disappears.

scholarly journals A role for I B kinase 2 in bipolar spindle assembly

2007 ◽  
Vol 104 (43) ◽  
pp. 16940-16945 ◽  
Author(s):  
J. T. Irelan ◽  
T. J. Murphy ◽  
P. D. DeJesus ◽  
H. Teo ◽  
D. Xu ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Bipolar Spindle

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.

The mitosis-regulating and protein-protein interaction activities of Astrin are controlled by Aurora-A-induced phosphorylation

2014 ◽  
Vol 307 (5) ◽  
pp. C466-C478 ◽  
Author(s):  
Shao-Chih Chiu ◽  
Jo-Mei Maureen Chen ◽  
Tong-You Wade Wei ◽  
Tai-Shan Cheng ◽  
Ya-Hui Candice Wang ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Spindle Assembly Checkpoint ◽  
Morphological Changes ◽  
Spindle Assembly ◽  
Aurora A ◽  
Protein Protein Interaction ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Complicated Process ◽  
Spindle Stability

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser115. The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

scholarly journals Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes.

1995 ◽  
Vol 131 (5) ◽  
pp. 1125-1131 ◽  
Author(s):  
D Zhang ◽  
R B Nicklas
Keyword(s):  
Nuclear Envelope ◽  
Essential Role ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Chromosomal Protein ◽  
Spindle Microtubule ◽  
Late Prophase ◽  
Spindle Formation ◽  
Bipolar Spindle

Chromosomes are known to enhance spindle microtubule assembly in grasshopper spermatocytes, which suggested to us that chromosomes might play an essential role in the initiation of spindle formation. Chromosomes might, for example, activate other spindle components such as centrosomes and tubulin subunits upon the breakdown of the nuclear envelope. We tested this possibility in living grasshopper spermatocytes. We ruptured the nuclear envelope during prophase, which prematurely exposed the centrosomes to chromosomes and nuclear sap. Spindle assembly was promptly initiated. In contrast, assembly of the spindle was completely inhibited if the nucleus was mechanically removed from a late prophase cell. Other experiments showed that the trigger for spindle assembly is associated with the chromosomes; other constituents of the nucleus cannot initiate spindle assembly in the absence of the chromosomes. The initiation of spindle assembly required centrosomes as well as chromosomes. Extracting centrosomes from late prophase cells completely inhibited spindle assembly after dissolution of the nuclear envelope. We conclude that the normal formation of a bipolar spindle in grasshopper spermatocytes is regulated by chromosomes. A possible explanation is an activator, perhaps a chromosomal protein (Yeo, J.-P., F. Alderuccio, and B.-H. Toh. 1994a. Nature (Lond.). 367: 288-291), that promotes and stabilizes the assembly of astral microtubules and thus promotes assembly of the spindle.

scholarly journals Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation

2015 ◽  
Vol 35 (15) ◽  
pp. 2626-2640 ◽  
Author(s):  
Lingjun Meng ◽  
Jung-Eun Park ◽  
Tae-Sung Kim ◽  
Eun Hye Lee ◽  
Suk-Youl Park ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Spindle Assembly ◽  
Human Cells ◽  
Mitotic Progression ◽  
Spindle Formation ◽  
Content Type ◽  
Bipolar Spindle ◽  
Egg Extracts ◽  
Cultured Human Cells ◽  
Bipolar Spindles

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study withXenopus laevisegg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif ofXenopusCep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous toXenopusp-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

scholarly journals Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Irsa Shoukat ◽  
Corey Frazer ◽  
John S. Allingham
Keyword(s):  
Candida Albicans ◽  
Mitotic Spindle ◽  
Fungal Pathogens ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Content Type ◽  
Bipolar Spindle ◽  
Spindle Elongation ◽  
Simultaneous Loss ◽  
Bipolar Spindles

ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.

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