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MPF localization is controlled by nuclear export
A Hagting1, C Karlsson, P Clute
1Wellcome/CRC Institute and Department of Zoology, Tennis Court Road, Cambridge, CB2 1QR, UK.
Abstract:
In eukaryotes, mitosis is initiated by M phase promoting factor (MPF), composed of B-type cyclins and their partner protein kinase, CDK1. In animal cells, MPF is cytoplasmic in interphase and is translocated into the nucleus after mitosis has begun, after which it associates with the mitotic apparatus until the cyclins are degraded in anaphase. We have used a fusion protein between human cyclin B1 and green fluorescent protein (GFP) to study this dynamic behaviour in real time, in living cells. We found that when we injected cyclin B1-GFP, or cyclin B1-GFP bound to CDK1 (i.e. MPF), into interphase nuclei it is rapidly exported into the cytoplasm. Cyclin B1 nuclear export is blocked by leptomycin B, an inhibitor of the recently identified export factor, exportin 1 (CRM1). The nuclear export of MPF is mediated by a nuclear export sequence in cyclin B1, and an export-defective cyclin B1 accumulates in interphase nuclei. Therefore, during interphase MPF constantly shuttles between the nucleus and the cytoplasm, but the bulk of MPF is retained in the cytoplasm by rapid nuclear export. We found that a cyclin mutant with a defective nuclear export signal does not enhance the premature mitosis caused by interfering with the regulatory phosphorylation of CDK1, but is more sensitive to inhibition by the Wee1 kinase.
Insights
M phase promoting factor (MPF) shuttles between the nucleus and cytoplasm in interphase animal cells. Rapid nuclear export retains most MPF in the cytoplasm, regulating cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitosis initiation in eukaryotes relies on M phase promoting factor (MPF), a complex of B-type cyclins and CDK1.
- In animal cells, MPF resides in the cytoplasm during interphase and translocates to the nucleus post-mitosis initiation.
Purpose of the Study:
- To investigate the dynamic behavior and localization of human cyclin B1 and MPF in living cells using real-time imaging.
- To elucidate the mechanisms governing MPF nuclear import and export during the cell cycle.
Main Methods:
- Utilized a fusion protein of human cyclin B1 and green fluorescent protein (GFP) for live-cell imaging.
- Injected cyclin B1-GFP and MPF into interphase nuclei and observed their localization.
- Employed leptomycin B to investigate the role of exportin 1 (CRM1) in nuclear export.
- Studied the effect of a nuclear export signal-defective cyclin B1 mutant.
Main Results:
- Injected cyclin B1-GFP and MPF were rapidly exported from the nucleus to the cytoplasm.
- Nuclear export of cyclin B1 was inhibited by leptomycin B, implicating exportin 1 (CRM1).
- A nuclear export sequence within cyclin B1 mediates MPF nuclear export, with defective mutants accumulating in interphase nuclei.
- MPF exhibits continuous nucleocytoplasmic shuttling, with rapid export maintaining cytoplasmic retention.
Conclusions:
- MPF localization is actively regulated by CRM1-mediated nuclear export during interphase.
- Defective nuclear export of cyclin B1 impacts MPF's role in cell cycle regulation, affecting sensitivity to CDK1 phosphorylation and Wee1 kinase inhibition.