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Updated: Dec 17, 2025
Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations
Published on: September 20, 2019
PP2A--B55γ counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA
Riya Keshri1, Ashwathi Rajeevan1, Sachin Kotak2
1Department of Microbiology and Cell Biology, Indian Institute of Science, 560012 Bangalore, India.
Abstract:
Proper orientation of the mitotic spindle is critical for accurate development and morphogenesis. In human cells, spindle orientation is regulated by the evolutionarily conserved protein NuMA, which interacts with dynein and enriches it at the cell cortex. Pulling forces generated by cortical dynein orient the mitotic spindle. Cdk1-mediated phosphorylation of NuMA at threonine 2055 (T2055) negatively regulates its cortical localization. Thus, only NuMA not phosphorylated at T2055 localizes at the cell cortex. However, the identity and the mechanism of action of the phosphatase complex involved in T2055 dephosphorylation remains elusive. Here, we characterized the PPP2CA-B55γ (PPP2R2C)-PPP2R1B complex that counteracts Cdk1 to orchestrate cortical NuMA for proper spindle orientation. In vitro reconstitution experiments revealed that this complex is sufficient for T2055 dephosphorylation. Importantly, we identified polybasic residues in NuMA that are critical for T2055 dephosphorylation, and for maintaining appropriate cortical NuMA levels for accurate spindle elongation. Furthermore, we found that Cdk1-mediated phosphorylation and PP2A-B55γ-mediated dephosphorylation at T2055 are reversible events. Altogether, this study uncovers a novel mechanism by which Cdk1 and its counteracting PP2A-B55γ complex orchestrate spatiotemporal levels of cortical force generators for flawless mitosis.
Insights
A phosphatase complex containing PPP2CA-B55γ counteracts Cdk1 to regulate NuMA
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitotic spindle orientation is crucial for cell division and development.
- NuMA protein localization at the cell cortex, regulated by dynein-mediated forces, controls spindle orientation.
- Cdk1 phosphorylation of NuMA at T2055 inhibits its cortical localization, but the dephosphorylation mechanism was unknown.
Purpose of the Study:
- To identify the phosphatase complex responsible for dephosphorylating NuMA at T2055.
- To elucidate the mechanism by which this phosphatase regulates NuMA's cortical localization and spindle orientation.
- To understand the interplay between Cdk1 phosphorylation and phosphatase-mediated dephosphorylation of NuMA.
Main Methods:
- Biochemical assays using purified proteins.
- In vitro reconstitution experiments.
- Identification of key residues in NuMA using mutagenesis.
Main Results:
- Characterization of the PPP2CA-B55γ-PPP2R1B complex as the phosphatase for NuMA T2055.
- Demonstration that this complex is sufficient for T2055 dephosphorylation in vitro.
- Identification of polybasic residues in NuMA critical for dephosphorylation and cortical localization.
- Evidence for reversible phosphorylation and dephosphorylation of NuMA at T2055.
Conclusions:
- A novel mechanism involving Cdk1 and PP2A-B55γ orchestrates NuMA's spatiotemporal cortical levels.
- This regulation is essential for accurate spindle orientation and flawless mitosis.
- The findings reveal a key regulatory axis controlling cortical force generators during cell division.
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