Nucleophosmin is required for chromosome congression, proper mitotic spindle formation, and kinetochore-microtubule attachment in HeLa cells

SUMMARYClathrin plays an important role to ensure mitotic spindle stability and efficient chromosome alignment, independently of its well-characterized functions in vesicle trafficking. While clathrin clearly localizes to the mitotic spindle and kinetochore-fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules have remained elusive. Here we show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the mitotic spindle. Structural analyses reveal that multiple clathrin-box motifs on GTSE1 interact directly with different clathrin adaptor interaction sites on CHC, in a manner structurally analogous to that which occurs between adaptor proteins and CHC near membranes. Specific disruption of this interaction in cells releases GTSE1 from spindles and causes defects in chromosome alignment. Surprisingly, this disruption causes destabilization of astral microtubules, but not kinetochore-microtubule attachments, and the resulting chromosome alignment defect is due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. Finally, we show that GTSE1 recruited to the spindle by clathrin stabilizes microtubules and promotes chromosome congression by inhibiting the activity of the microtubule depolymerase MCAK. This work thus uncovers a novel role of clathrin to stabilize non-kinetochore-fiber microtubules to support chromosome congression. This role is carried out via clathrin adaptor-type interactions of CHC with GTSE1, defining for the first time an important repurposing of this endocytic interaction mechanism during mitosis.

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Aurora-A kinase-inactive mutants disrupt the interaction with Ajuba and cause defects in mitotic spindle formation and G2/M phase arrest in HeLa cells

BMB Reports ◽

10.5483/bmbrep.2014.47.11.250 ◽

2014 ◽

Vol 47 (11) ◽

pp. 631-636 ◽

Cited By ~ 2

Author(s):

Meirong Bai ◽

Jun Ni ◽

Suqin Shen ◽

Qiang Huang ◽

Jiaxue Wu ◽

...

Keyword(s):

Hela Cells ◽

Mitotic Spindle ◽

Aurora A Kinase ◽

Aurora A ◽

Spindle Formation ◽

Phase Arrest ◽

M Phase

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MKLP2 functions in early mitosis to ensure proper chromosome congression

10.1101/2021.09.12.459884 ◽

2021 ◽

Author(s):

Morgan S Schrock ◽

Luke Scarberry ◽

Benjamin R Stromberg ◽

Claire Sears ◽

Adrian E Torres ◽

...

Keyword(s):

Error Correction ◽

Small Molecule ◽

Hela Cells ◽

Chromosomal Instability ◽

Direct Evidence ◽

Aurora Kinase ◽

Downstream Target ◽

Microtubule Attachment ◽

Chromosome Congression ◽

Early Mitosis

Mitotic kinesin-like protein 2 (MKLP2) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small molecule inhibitors of MKLP2 (MKLP2i) along with the observations of others suggested a function earlier in mitosis, prior to anaphase. In this study we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 inhibited cells had a significant increase in unstable kinetochore-microtubule attachments due to impaired error correction of syntelic attachments. We propose that MKLP2 mediates kinetochore microtubule attachment stability by regulating Aurora Kinase and a downstream target, pHEC1 (Ser 55). Lastly, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability.

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Centriole-independent mitotic spindle assembly relies on the PCNT–CDK5RAP2 pericentriolar matrix

The Journal of Cell Biology ◽

10.1083/jcb.202006010 ◽

2020 ◽

Vol 219 (12) ◽

Author(s):

Sadanori Watanabe ◽

Franz Meitinger ◽

Andrew K. Shiau ◽

Karen Oegema ◽

Arshad Desai

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Hela Cells ◽

Mitotic Spindle ◽

Spindle Assembly ◽

Cell Type ◽

Matrix Assembly ◽

Spindle Formation ◽

Pericentriolar Material ◽

Mitotic Spindle Assembly

Centrosomes, composed of centrioles that recruit a pericentriolar material (PCM) matrix assembled from PCNT and CDK5RAP2, catalyze mitotic spindle assembly. Here, we inhibit centriole formation and/or remove PCNT–CDK5RAP2 in RPE1 cells to address their relative contributions to spindle formation. While CDK5RAP2 and PCNT are normally dispensable for spindle formation, they become essential when centrioles are absent. Acentriolar spindle assembly is accompanied by the formation of foci containing PCNT and CDK5RAP2 via a microtubule and Polo-like kinase 1–dependent process. Foci formation and spindle assembly require PCNT-CDK5RAP2–dependent matrix assembly and the ability of CDK5RAP2 to recruit γ-tubulin complexes. Thus, the PCM matrix can self-organize independently of centrioles to generate microtubules for spindle assembly; conversely, an alternative centriole-anchored mechanism supports spindle assembly when the PCM matrix is absent. Extension to three cancer cell lines revealed similar results in HeLa cells, whereas DLD1 and U2OS cells could assemble spindles in the absence of centrioles and PCNT-CDK5RAP2, suggesting cell type variation in spindle assembly mechanisms.

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Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

The Journal of Cell Biology ◽

10.1083/jcb.201907083 ◽

2020 ◽

Vol 219 (2) ◽

Cited By ~ 8

Author(s):

Arnaud Rondelet ◽

Yu-Chih Lin ◽

Divya Singh ◽

Arthur T. Porfetye ◽

Harish C. Thakur ◽

...

Keyword(s):

Mitotic Spindle ◽

Interaction Mechanism ◽

Interaction Sites ◽

Microtubule Attachment ◽

Chromosome Congression ◽

Chromosome Alignment ◽

Clathrin Adaptor ◽

Spindle Stability ◽

First Time

Clathrin ensures mitotic spindle stability and efficient chromosome alignment, independently of its vesicle trafficking function. Although clathrin localizes to the mitotic spindle and kinetochore fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules are unclear. We show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the spindle. Structural analyses reveal that these sites interact directly with clathrin-box motifs on GTSE1. Disruption of this interaction releases GTSE1 from spindles, causing defects in chromosome alignment. Surprisingly, this disruption destabilizes astral microtubules, but not kinetochore-microtubule attachments, and chromosome alignment defects are due to a failure of chromosome congression independent of kinetochore–microtubule attachment stability. GTSE1 recruited to the spindle by clathrin stabilizes microtubules by inhibiting the microtubule depolymerase MCAK. This work uncovers a novel role of clathrin adaptor-type interactions to stabilize nonkinetochore fiber microtubules to support chromosome congression, defining for the first time a repurposing of this endocytic interaction mechanism during mitosis.

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