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Updated: Nov 9, 2025
Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations
Published on: September 20, 2019
Spatiotemporal coordination of Greatwall-Endos-PP2A promotes mitotic progression
Myreille Larouche1,2, David Kachaner1,2, Peng Wang1,2
1Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec, Canada.
Abstract:
Mitotic entry involves inhibition of protein phosphatase 2A bound to its B55/Tws regulatory subunit (PP2A-B55/Tws), which dephosphorylates substrates of mitotic kinases. This inhibition is induced when Greatwall phosphorylates Endos, turning it into an inhibitor of PP2A-Tws. How this mechanism operates spatiotemporally in the cell is incompletely understood. We previously reported that the nuclear export of Greatwall in prophase promotes mitotic progression. Here, we examine the importance of the localized activities of PP2A-Tws and Endos for mitotic regulation. We find that Tws shuttles through the nucleus via a conserved nuclear localization signal (NLS), but expression of Tws in the cytoplasm and not in the nucleus rescues the development of tws mutants. Moreover, we show that Endos must be in the cytoplasm before nuclear envelope breakdown (NEBD) to be efficiently phosphorylated by Greatwall and to bind and inhibit PP2A-Tws. Disrupting the cytoplasmic function of Endos before NEBD results in subsequent mitotic defects. Evidence suggests that this spatiotemporal regulation is conserved in humans.
Insights
Cytoplasmic localization of Endos and PP2A-Tws is crucial for timely mitotic entry. This spatiotemporal regulation ensures proper inhibition of PP2A-Tws by Greatwall, preventing mitotic defects.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Mitotic entry requires precise regulation of protein dephosphorylation.
- Protein phosphatase 2A bound to its B55/Tws regulatory subunit (PP2A-Tws) dephosphorylates mitotic kinase substrates.
- Inhibition of PP2A-Tws is mediated by Greatwall-phosphorylated Endos.
Purpose of the Study:
- To investigate the spatiotemporal regulation of PP2A-Tws and Endos during mitosis.
- To understand how localized activities of these proteins impact mitotic progression.
- To elucidate the role of Endos's cytoplasmic localization before nuclear envelope breakdown.
Main Methods:
- Analysis of Tws nuclear shuttling via its nuclear localization signal (NLS).
- Functional rescue experiments of tws mutants by cytoplasmic vs. nuclear Tws expression.
- Investigating the timing and localization of Endos phosphorylation by Greatwall.
- Assessing mitotic defects upon disruption of Endos's cytoplasmic function before NEBD.
Main Results:
- Tws shuttles between the nucleus and cytoplasm, but only cytoplasmic Tws rescues tws mutant phenotypes.
- Cytoplasmic localization of Endos before NEBD is essential for its phosphorylation by Greatwall.
- Endos must be cytoplasmic before NEBD to effectively bind and inhibit PP2A-Tws.
- Disrupting Endos's cytoplasmic function prior to NEBD leads to mitotic abnormalities.
Conclusions:
- The spatiotemporal control of Endos and PP2A-Tws localization is critical for regulating mitotic entry.
- Cytoplasmic localization of Endos before NEBD is a key regulatory step.
- This mechanism of mitotic regulation appears conserved in humans.
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