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PML bodies control the nuclear dynamics and function of the CHFR mitotic checkpoint protein

Nature Structural & Molecular Biology volume 11, pages 1114–1121 (2004)Cite this article

Abstract

Nuclear foci containing the promyelocytic leukemia protein (PML bodies), which occur in most cells, play a role in tumor suppression. Here, we demonstrate that CHFR, a mitotic checkpoint protein frequently inactivated in human cancers, is a dynamic component of PML bodies. Intermolecular fluorescence resonance energy transfer analysis identified a distinct fraction of CHFR that interacts with PML in living cells. This interaction modulates the nuclear distribution and mobility of CHFR. A trans-dominant mutant of CHFR that inhibits checkpoint function also prevents colocalization and interaction with PML. Conversely, the distribution and mobility of CHFR are perturbed in PML−/− cells, accompanied by aberrations in mitotic entry and the response to spindle depolymerization. Thus, PML bodies control the distribution, dynamics and function of CHFR. Our findings implicate the interaction between these tumor suppressors in a checkpoint response to microtubule poisons, an important class of anticancer drugs.

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Figure 1: CHFR and PML colocalize and interact.
Figure 2: Intermolecular FRET between CFP-CHFR and PML-YFP.
Figure 3: Intermolecular FRET between CFP-CHFR and PML-YFP in living cells.
Figure 4: Movement of small GFP-CHFR-containing foci to and from PML bodies.
Figure 5: CHFRΔFHA prevents recruitment of CHFR, but not of PML, into PML bodies.
Figure 6: Control of CHFR dynamics in PML bodies.
Figure 7: Perturbed CHFR localization and mitotic checkpoint abnormalities after PML disruption.

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Acknowledgements

M. Daniels generously assisted us with XR1 cell culture and preparation of X. laevis extracts, and T. Mills with microinjection. T. Rich (Department of Pathology, University of Cambridge) and P.-P. Pandolfi (Memorial Sloan-Kettering Institute, New York) supplied the PML-YFP construct, and PML−/− and control MEFs, respectively, and provided constructive comments on this paper, as did L. Ko-Ferrigno. M.J.D. was supported by an Astra-Zeneca studentship, through the MB/PhD program at the University of Cambridge, and by the UK Medical Research Council. A.M. was the receipient of a Herchel Smith Harvard scholarship. Work in A.R.V.'s laboratory is supported by the Medical Research Council and Cancer Research UK.

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  1. Cancer Research UK Department of Oncology and The Medical Research Council Cancer Cell Unit, University of Cambridge, Hills Road, Cambridge, CB2 2XZ, UK

    Matthew J Daniels, Alexander Marson & Ashok R Venkitaraman

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Correspondence to Ashok R Venkitaraman.

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Supplementary information

Supplementary Fig. 1

Negative control experiments for FRET interaction between CFP-CHFR and PML-YFP. (PDF 1505 kb)

Supplementary Fig. 2

GFP does not coprecipitate with PML. (PDF 635 kb)

Supplementary Video 1

Two-dimensional rendering of serial z-sections. To exclude the possibility that the small CHFR-containing foci shown in Figure 4 are migrating in/out of the plane-of-focus, we acquired images of an entire YFP-CHFR-expressing cell nucleus with overlapping z-sections over time. We have then rendered the three-dimensional image at each time point into two dimensions as a maximum intensity projection. When played in sequence, this series of images reveals the movement of all YFP-CHFR-containing foci within a single cell nucleus. The Supplementary Video file shows this experiment. It suggests that most of the CHFR-containing foci are not simply migrating in/out of the plane of focus, but are indeed interacting with large foci as discussed in the main text. (AVI 35101 kb)

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Daniels, M., Marson, A. & Venkitaraman, A. PML bodies control the nuclear dynamics and function of the CHFR mitotic checkpoint protein. Nat Struct Mol Biol 11, 1114–1121 (2004). https://doi.org/10.1038/nsmb837

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