scholarly journals The association of Plk1 with the Astrin-Kinastrin complex promotes formation and maintenance of a metaphase plate

2020 ◽  
Author(s):  
Zoë Geraghty ◽  
Christina Barnard ◽  
Pelin Uluocak ◽  
Ulrike Gruneberg
Keyword(s):  
Molecular Mechanisms ◽  
Metaphase Plate ◽  
Direct Interaction ◽  
Mitotic Chromosomes ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment

AbstractErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The Astrin-Kinastrin/SKAP complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule-kinetochore attachment. However, the molecular mechanisms by which Astrin-Kinastrin fulfils these diverse roles are not fully understood. Here we characterise a direct interaction between Astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on Astrin as well as four Plk1 phosphorylation sites on Astrin. Regulation of Astrin-Kinastrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression but promotes stable microtubule-kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule-kinetochore attachment formation, and we suggest that Astrin phosphorylation by Plk1 contributes to this process.SummaryWe demonstrate that Plk1 binds to and phosphorylates the N-terminus of Astrin. This interaction promotes recruitment of the Astrin-complex to kinetochores and stabilises microtubule-kinetochore-attachments in situations when mitosis is delayed.

scholarly journals The association of Plk1 with the astrin–kinastrin complex promotes formation and maintenance of a metaphase plate

2020 ◽  
Vol 134 (1) ◽  
pp. jcs251025
Author(s):  
Zoë Geraghty ◽  
Christina Barnard ◽  
Pelin Uluocak ◽  
Ulrike Gruneberg
Keyword(s):  
Molecular Mechanisms ◽  
Metaphase Plate ◽  
Direct Interaction ◽  
Mitotic Chromosomes ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment

ABSTRACTErrors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin–kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule–kinetochore attachment. However, the molecular mechanisms by which astrin–kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule–kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule–kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

scholarly journals Kif18A promotes Hec1 dephosphorylation to coordinate chromosome alignment with kinetochore microtubule attachment

2018 ◽  
Author(s):  
Haein Kim ◽  
Jason Stumpff
Keyword(s):  
Molecular Mechanisms ◽  
Spindle Assembly Checkpoint ◽  
Primordial Germ Cells ◽  
Direct Interaction ◽  
Mitotic Arrest ◽  
Microtubule Dynamics ◽  
Mitotic Chromosomes ◽  
Microtubule Attachment ◽  
C Terminus ◽  
Chromosome Alignment

SUMMARYMitotic chromosomes are spatially confined at the spindle equator just prior to chromosome segregation through a process called chromosome alignment. Alignment requires temporal coordination of kinetochore microtubule attachment and dynamics. However, the molecular mechanisms that couple these activities are not understood. Kif18A (kinesin-8) suppresses the dynamics of kinetochore microtubules to promote chromosome alignment during metaphase. Loss of Kif18A function in HeLa and primordial germ cells leads to alignment defects accompanied by a spindle assembly checkpoint (SAC)-dependent mitotic arrest, suggesting the motor also plays a role in regulating kinetochore-microtubule attachments. We show here that Kif18A increases attachment by promoting dephosphorylation of the kinetochore protein Hec1, which provides the primary linkage between kinetochores and microtubules. This function requires a direct interaction between the Kif18A C-terminus and protein phosphatase-1 (PP1). However, the Kif18A-PP1 interaction is not required for chromosome alignment, indicating that regulation of kinetochore microtubule dynamics and attachments are separable Kif18A functions. Mitotic arrest in Kif18A-depleted cells is rescued by expression of a Hec1 variant that mimics a low-phosphorylation state, indicating that Kif18A-dependent Hec1 dephosphorylation is a key step for silencing the checkpoint and promoting mitotic progression. Our data support a model in which Kif18A provides positive feedback for kinetochore microtubule attachment by directly recruiting PP1 to dephosphorylate Hec1. We propose that this function works synergistically with Kif18A’s direct control of kinetochore microtubule dynamics to temporally coordinate chromosome alignment and attachment.

scholarly journals Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression and alignment during mitosis

2021 ◽  
Vol 118 (34) ◽  
pp. e2108145118
Author(s):  
Anja Bufe ◽  
Ana García del Arco ◽  
Magdalena Hennecke ◽  
Anchel de Jaime-Soguero ◽  
Matthias Ostermaier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Division ◽  
Wnt Signaling ◽  
Pluripotent Stem Cells ◽  
Target Genes ◽  
Genome Maintenance ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Canonical Wnt

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating β-catenin target genes. Recent evidence showed that β-catenin–independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.

scholarly journals Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

2021 ◽  
Vol 220 (2) ◽  
Author(s):  
Takumi Chinen ◽  
Kaho Yamazaki ◽  
Kaho Hashimoto ◽  
Ken Fujii ◽  
Koki Watanabe ◽  
...  
Keyword(s):  
A549 Cells ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Scaffold Proteins ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Mitotic Cells

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

scholarly journals Elevated polar ejection forces stabilize kinetochore–microtubule attachments

2013 ◽  
Vol 200 (2) ◽  
pp. 203-218 ◽  
Author(s):  
Stuart Cane ◽  
Anna A. Ye ◽  
Sasha J. Luks-Morgan ◽  
Thomas J. Maresca
Keyword(s):  
Molecular Motors ◽  
Molecular Mechanisms ◽  
Critical Force ◽  
Aurora B ◽  
Dependent Manner ◽  
Spindle Poles ◽  
Ejection Force ◽  
Chromosome Alignment ◽  
Directed Motility ◽  
Spindle Microtubules

Chromosome biorientation promotes congression and generates tension that stabilizes kinetochore–microtubule (kt-MT) interactions. Forces produced by molecular motors also contribute to chromosome alignment, but their impact on kt-MT attachment stability is unclear. A critical force that acts on chromosomes is the kinesin-10–dependent polar ejection force (PEF). PEFs are proposed to facilitate congression by pushing chromosomes away from spindle poles, although knowledge of the molecular mechanisms underpinning PEF generation is incomplete. Here, we describe a live-cell PEF assay in which tension was applied to chromosomes by manipulating levels of the chromokinesin NOD (no distributive disjunction; Drosophila melanogaster kinesin-10). NOD stabilized syntelic kt-MT attachments in a dose- and motor-dependent manner by overwhelming the ability of Aurora B to mediate error correction. NOD-coated chromatin stretched away from the pole via lateral and end-on interactions with microtubules, and NOD chimeras with either plus end–directed motility or tip-tracking activity produced PEFs. Thus, kt-MT attachment stability is modulated by PEFs, which can be generated by distinct force-producing interactions between chromosomes and dynamic spindle microtubules.

scholarly journals Clathrin recruits phosphorylated TACC3 to spindle poles for bipolar spindle assembly and chromosome alignment

2010 ◽  
Vol 123 (21) ◽  
pp. 3645-3651 ◽  
Author(s):  
W. Fu ◽  
W. Tao ◽  
P. Zheng ◽  
J. Fu ◽  
M. Bian ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Chromosome Alignment

scholarly journals Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

2020 ◽  
Author(s):  
Takumi Chinen ◽  
Kaho Yamazaki ◽  
Kaho Hashimoto ◽  
Ken Fujii ◽  
Koki Watanabe ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Spindle Pole ◽  
Human Cells ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
The Individual ◽  
Chemical Perturbation

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. While centrosomes facilitate bipolar spindle formation, the individual functions of the centriole and PCM in mitosis remain elusive. Herein, we show the redundant roles of the centriole and PCM in bipolar spindle formation in human cells. Upon depletion of the PCM scaffold components, pericentrin and CDK5RAP2, centrioles remained able to recruit CEP192 onto their walls, which was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the chemical perturbation of polo-like kinase 1, a critical kinase for PCM assembly, efficiently suppressed the proliferation of various cancer cell lines from which centrioles were removed. Overall, these data suggest that the centriole and PCM cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation in human cells.

scholarly journals More than Two Populations of Microtubules Comprise the Dynamic Mitotic Spindle

2021 ◽  
Author(s):  
Aaron R. Tipton ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Metaphase Plate ◽  
Spindle Poles ◽  
Chromatid Segregation ◽  
Chromosome Alignment ◽  
Chromosome Attachment ◽  
Complex Landscape ◽  
Two Populations ◽  
Insight Into ◽  
Slow Turnover

The microtubules of the mitotic spindle mediate chromosome alignment to the metaphase plate, then sister chromatid segregation to the spindle poles in anaphase. Previous analyses of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, and these were ascribed to reflect kinetochore versus non-kinetochore microtubules, respectively. Here, we test this categorization by disrupting kinetochores through depletion of the Ndc80 complex. In the absence of functional kinetochores, microtubule dynamics still exhibit slow and fast turnover populations, though the proportion of each population and the timings of turnover are altered. Importantly, the data obtained following Hec1/Ndc80 depletion suggests other sub-populations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. For example, while Aurora B kinase functions to destabilize kinetochore bound microtubules it may also stabilize certain slow turnover, non-kinetochore microtubules. Dissection of the dynamics of microtubule populations provides a greater understanding of mitotic spindle kinetics and insight into their roles in facilitating chromosome attachment, movement, and segregation during mitosis.

scholarly journals CENP-E Function at Kinetochores Is Essential for Chromosome Alignment

1997 ◽  
Vol 139 (6) ◽  
pp. 1373-1382 ◽  
Author(s):  
B.T. Schaar ◽  
G.K.T. Chan ◽  
P. Maddox ◽  
E.D. Salmon ◽  
T.J. Yen
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Metaphase Plate ◽  
Motor Domain ◽  
Amino Terminal ◽  
Spindle Poles ◽  
Opposite Pole ◽  
Chromosome Alignment ◽  
Combined Data

CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions that are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies to block the assembly of CENP-E onto kinetochores and then examined the behavior of these chromosomes. Chromosomes lacking CENP-E at their kinetochores consistently exhibited two types of defects that blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they were unable to establish bipolar microtubule connections with the opposite pole. Chromosomes within the spindle established bipolar connections that supported oscillations and normal velocities of kinetochore movement between the poles, but these bipolar connections were defective because they failed to align the chromosomes into a metaphase plate. Overexpression of a mutant that lacked the amino-terminal 803 amino acids of CENP-E was found to saturate limiting binding sites on kinetochores and competitively blocked endogenous CENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be aligned but accumulated at the poles or were strewn within the spindle as was the case when cells were microinjected with CENP-E antibodies. As the motor domain was contained within the portion of CENP-E that was deleted, the chromosomal defect is likely attributed to the loss of motor function. The combined data show that CENP-E provides kinetochore functions that are essential for monopolar chromosomes to establish bipolar connections and for chromosomes with connections to both spindle poles to align at the spindle equator. Both of these events rely on activities that are provided by CENP-E's motor domain.

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