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The PML protein is a target of the t(15;17) chromosomal translocationtypical of promyelocytic variants of acute myeloid leukemia, whichfuses PML reciprocally with retinoic acid receptor α (RARα)1.Overexpression of PML in cancer cell lines induces growth arrest andapoptosis2. PML-RARα fusion proteins that interfere with the func-tion of endogenous PML in a trans-dominant manner1,3 causeleukemia in transgenic mice, whereas PML–/– mice develop a range ofcancers including papillomas, carcinomas and lymphomas after expo-sure to carcinogens4; this is paralleled by the loss of PML in humancancers from diverse tissues5. Thus, PML is a potent tumor suppressorin vitro and in vivo, in many cell types.

PML accumulates in focal nuclear structures termed PML bodies2,with an average size of ∼ 1 µm, which vary in number from 10–30 pernucleus. PML bodies are not formed in PML–/– cells4,6, or in cells thatexpress PML-RARα fusions1. Their disappearance is correlated withabnormalities in cell growth and death through effects on transcrip-tional regulation, as well as p53-dependent and independent apopto-sis7. Because PML bodies contain several different molecules2,including the proapoptotic factor DAXX and the tumor suppressorsp53, Rb, Chk2 and BRCA1, it has been proposed that they may serve asrepositories for protein storage or modification8. However, the vari-able movement of proteins to and from PML bodies predicted by thishypothesis has yet to be directly demonstrated.

We investigated the intracellular trafficking of the checkpoint proteinCHFR and report here its hitherto unrecognized interaction with PMLbodies. CHFR, a RING- and FHA-domain-containing nuclear protein, isinactivated by epigenetic mechanisms in up to 30% of epithelial cancercell lines9. CHFR inactivation abrogates the transient delay in passagethrough early mitotic prophase induced by microtubule-disruptingagents9. We find that the nuclear distribution and mobility of CHFR

depend on its movement through PML bodies, revealing novel aspects oftheir putative role as depots for nuclear proteins. This regulation is per-turbed by a trans-dominant inhibitory mutant of CHFR or in PML–/–

cells, and is accompanied by abnormalities in mitotic progression and theresponse to microtubule depolymerization. Our findings identify a novelinteraction between two tumor suppressor proteins that provides insightinto the biology of PML bodies, and has implications for cancer therapy.

RESULTSAlterations in GFP-CHFR distribution during the cell cycleGreen fluorescent protein (GFP) was fused in-frame to the N terminusof human CHFR (Fig. 1a) to yield a GFP-CHFR fusion protein with apredicted molecular mass of ∼ 110 kDa (Fig. 1b). A similar GFP-CHFRfusion protein has been used previously, and can reconstitute functionin CHFR-deficient cells10. We used it to examine the intracellular dis-tribution of CHFR, because antibodies for fluorescent staining of theendogenous human protein have not yet been reported, nor have webeen able to raise them. To provide evidence that GFP-CHFR behavessimilarly to the endogenous molecule, we cloned the CHFR orthologfrom Xenopus laevis (xCHFR), and raised an antibody against it thatdetects a single band of the appropriate size in cell extracts (Fig. 1c).Staining of endogenous xCHFR in the XR1 cell line during interphase(Fig. 1d) revealed a punctate pattern of nuclear distribution very simi-lar to GFP-CHFR in HeLa cells (Fig. 1e). Moreover, endogenous x-CHFR and transfected GFP-CHFR colocalized extensively in XR1cells (Fig. 1d). Collectively, these findings provide evidence that GFP-CHFR accurately reflects the behavior of endogenous CHFR.

In HeLa cells during interphase, GFP-CHFR accumulates, primarilyin large and small focal structures that are excluded from the nucleolus,with a small amount distributed through the nucleoplasm. Coincident

University of Cambridge, Cancer Research UK Department of Oncology and The Medical Research Council Cancer Cell Unit, Hills Road, Cambridge CB2 2XZ, UK.Correspondence should be addressed to A.R.V. (arv22@cam.ac.uk).

Published online 3 October 2004; doi:10.1038/nsmb837

PML bodies control the nuclear dynamics and function ofthe CHFR mitotic checkpoint proteinMatthew J Daniels, Alexander Marson & Ashok R Venkitaraman

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 ofPML bodies. Intermolecular fluorescence resonance energy transfer analysis identified a distinct fraction of CHFR that interacts withPML in living cells. This interaction modulates the nuclear distribution and mobility of CHFR. A trans-dominant mutant of CHFR thatinhibits checkpoint function also prevents colocalization and interaction with PML. Conversely, the distribution and mobility of CHFRare perturbed in PML–/– cells, accompanied by aberrations in mitotic entry and the response to spindle depolymerization. Thus, PMLbodies control the distribution, dynamics and function of CHFR. Our findings implicate the interaction between these tumorsuppressors in a checkpoint response to microtubule poisons, an important class of anticancer drugs.

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with mitotic prophase, this pattern changes (Fig. 1e). Many of the focicontaining GFP-CHFR disperse, and the diffusely distributed proteinsurrounds but is excluded from the chromosome territories. A few smallfoci persist over chromatin. This mixed pattern continues after nuclearenvelope breakdown and the progressive condensation of chromosomesduring the remainder of mitosis. Similar results were obtained in SaOS2,HCT116 and 293T cells. So, the intracellular distribution of GFP-CHFRis regulated during progression through the cell cycle.

Large GFP-CHFR foci coincide with PML bodiesGFP-CHFR foci in interphase cells range in size (Fig. 1f) from small(average diameter 0.31 ± 0.065 µm) to large (average diameter 0.77 ± 0.1µm). Small foci occur (Fig. 1g) in about four-fold greater numbers (largefoci, 6.9 ± 4.4 (range 0–13) per nucleus; small foci, 34 ± 9.4 (range 25–51)per nucleus). Similarities in the size and distribution of the large foci withthe reported characteristics of PML bodies11 prompted us to examine thecolocalization of the PML and CHFR proteins (Fig. 1h). EndogenousPML bodies revealed with an antibody against PML coincide with thelarge GFP-CHFR foci. Moreover, the SUMO-modified form of PML,which occurs in PML bodies and is required for their organization12,preferentially coimmunoprecipitates with GFP-CHFR (Fig. 1i).

Interaction of CHFR with PML by intermolecular FRETWe established a method for intermolecular fluorescence resonanceenergy transfer (FRET) to investigate the physical interaction between

CHFR and PML in living cells (Fig. 2a). FRET is the radiationless trans-fer of energy between a pair of suitably oriented, spectrally overlappingfluorophores in close physical proximity13. During FRET, the donorfluorophore’s emission is quenched whereas the acceptor fluorophore’semission is stimulated. In our experiments, a CFP-CHFR fusion pro-tein (whose intracellular distribution is indistinguishable from GFP-CHFR) acted as the FRET donor. The FRET acceptor was a PML-YFPfusion. Negative control experiments using cells transfected with a single fluorophore are shown in Supplementary Figure 1 online.

Intermolecular FRET between CFP-CHFR and PML-YFP wasdemonstrated first in fixed interphase HeLa cells using 405-nm light,which was sufficient only to excite CFP. In the CFP-CHFR prebleachimage in Figure 2b, large CHFR-containing foci are not visible,although small foci are clearly seen. By contrast, large foci are clearlyvisible in the PML-YFP image but small foci are not. In other words,donor (CFP) quenching and sensitized acceptor (YFP) emissionsoccur only in the large foci, demonstrating that FRET between CFP-CHFR and PML-YFP occurs exclusively in these structures—that is,in PML bodies.

To confirm the specificity of intermolecular FRET, PML-YFP wasirreversibly photobleached (Fig. 2c) before excitation of CFP-CHFRwith 405-nm light. Here we observed the reverse of what is seen inFigure 2b. CFP-CHFR is clearly visible in the large foci (PML bodies)within the photobleached area, but PML-YFP is not. The loss of donorquenching and of sensitized acceptor emissions after photobleachingconfirms the authenticity of FRET.

The endpoint of FRET imaging13, a ratio of donor emissionsbefore and after acceptor photobleaching, was rendered as apseudo-colored image (Fig. 2d). FRET ratios of ∼ 6.0 occur withinthe large foci (PML bodies) where CFP-CHFR and PML-YFP are inclose physical proximity. Thin peripheral rims with a lower FRETratio border the PML bodies, representing regions in which

Figure 1 CHFR and PML colocalize and interact. (a) Schematic of the GFP-CHFR fusion protein highlighting the FHA, RING-finger and cysteine-rich (CR)domains. (b) An anti-GFP western blot of extracts from HeLa cells expressingthe fusion proteins used here. The GFP CHFR∆FHA protein (relative molecularmass (Mr), 85 kDa) lacks the first 111 residues of CHFR. Lane 1, GFP-CHFR;lane 2, GFP-CHFR∆FHA; lane 3, PML-YFP. (c) Specificity of the antibodyagainst X. laevis CHFR (X-CHFR). The antibody was used to detect X-CHFR bywestern blotting in control reticulocyte lysates without added cDNA (lane 1),in vitro–translated X-CHFR cDNA (lane 2), or oocyte extracts (lane 3). (d) Localization of endogenous X-CHFR in foci, and colocalization withtransfected GFP-CHFR, in interphase XR1 cells. Results shown are typical of>50 cells microinjected with a plasmid encoding GFP-CHFR. Images aresuperimposed in the merge (DNA, anti-X-CHFR, GFP-CHFR) and two-pixelquantitative colocalization (anti-X-CHFR, GFP-CHFR) panels, with DNA inblue, anti-X-CHFR staining in red and GFP-CHFR in green. Areas ofcolocalization are yellow-green. Scale bar, 5 µm. (e) Cell cycle–dependentlocalization of GFP-CHFR in HeLa cells. GFP-CHFR is green, DNA stained withDAPI is blue, and kinetochores visualized with CREST antibody are red. Scalebar, 5 µm. (f) Localization of GFP-CHFR in interphase counterstained withDAPI (blue). Localization in large foci, small foci and diffuse staining in thenucleoplasm are visible. Scale bar, 5 µm. (g) Numbers of large and small fociwere enumerated per cell (n = 50). (h) Colocalization of GFP-CHFR withendogenous PML. Cells transfected with GFP-CHFR (green) were fixed andcounterstained with anti-PML (red). Colocalization in large foci (PML bodies)appears yellow in the merge image. Scale bar, 5 µm. (i) Anti-GFP or anti-PMLimmunoprecipitates from GFP-CHFR-transfected cells were blotted withmonoclonal anti-PML. GFP-CHFR preferentially associates with the PML bandcorresponding to the predicted migration of PML-(SUMO)3. Anti-PMLimmunoprecipitates contain multiple PML-SUMO isoforms. As a negativecontrol, the same immunoprecipitations were done using cells transfected withGFP alone (Supplementary Fig. 2 online).

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decreased FRET interaction might arise as a consequence of alteredintermolecular orientation, displacement or stoichiometry at theperiphery of the PML body.

Our observations reveal a novel feature of the organization of PMLbodies. These structures are not homogenous in terms of the interac-tion between CFP-CHFR and PML-YFP. Instead, they consist of acentral core and peripheral rim, which are demarcated as adjacentareas with different FRET ratios. This illustrates an application ofFRET imaging for the interrogation of subcellular structures withhigh resolution.

Intermolecular FRET in living cellsThese results were validated and extended using intermolecular FRETin unfixed, living HeLa cells. Before acceptor photobleaching (Fig. 3a),quenching of the CFP-CHFR donor rendered large foci (PML bodies)less visible, whereas sensitized acceptor emissions through FRET madethese structures visible in the adjacent PML-YFP image. This wasreversed after acceptor photobleaching in a single large focus (PMLbody) (Fig. 3b), confirming that specific intermolecular FRET occursin these structures. The heterogeneity in the interaction between CFP-

CHFR and PML-YFP is not so apparent in the pseudo-colored image(Fig. 3c). Indeed, the apparent FRET ratio is considerably lower thanin fixed cells, because of limitations imposed in living cells for imagingthis FRET pair.

Traffic of GFP-CHFR to and from PML bodiesSeveral lines of evidence (Figs. 1f,g, 2d and 3c) establish that a physi-cal interaction between CHFR and PML occurs only in large foci(PML bodies). But CHFR also accumulates in small nuclear foci dis-tinct from PML bodies. To define the relationship between thesestructures, we carried out three-dimensional reconstructive imagingof GFP-CHFR and endogenous PML (stained with anti-PML) infixed HeLa cells. Approximately 60 images were take through a singlecell (sufficient oversampling to ensure that each pixel was recordedin at least four focal z-planes), and the resultingimage set projectedas a single three-dimensional composite (Fig. 4a).

Figure 2 Intermolecular FRET between CFP-CHFR and PML-YFP. (a) Aschematic of intermolecular FRET. CFP excited with 405-nm light usuallyemits at 470 nm (no FRET, left). If correctly oriented CFP and YFPfluorophores are within nanometer proximity, FRET occurs, quenching theusual 470-nm emission from CFP, but exciting 535-nm emission from YFP(FRET, right). Note that 405-nm light cannot directly excite YFP(Supplementary Fig. 1 online). (b) Cells cotransfected with CFP-CHFR andPML-YFP were fixed and visualized using 405-nm laser light. Emissionspectra for CFP (green) and YFP (red) are shown, as is the merged image.Scale bar, 5 µm. (c) YFP was irreversibly photobleached with 514-nm lightin the areas bound by the white line, before visualization of CFP and YFPemission excited by 405-nm light. Scale bar, 5 µm. (d) A pseudo-coloredFRET image (right) shows the ratio of donor (CFP) emissions before (left)and after (middle) YFP photobleaching, according to ref. 12. Scale bar, 5 µm. The pseudo color scale marks the FRET ratio from 0 (blue) to 6.0(red). The image depicts the magnitude of interaction between donor andacceptor fluorophores within PML bodies, and also reveals the geography ofthat interaction as heterogeneities in the pseudo-colored FRET ratios.Results are typical of at least three independent experiments.

Figure 3 Intermolecular FRET between CFP-CHFR and PML-YFP in livingcells. (a) Cells cotransfected with CFP-CHFR and PML-YFP were visualizedusing 405-nm laser light. Emission spectra for CFP (green) and YFP (red)are shown, as is the merged image. Scale bar, 5 µm. (b) 514-nm light wasused to photobleach a single PML-YFP focus (white ring), beforevisualization of CFP and YFP emissions excited by 405-nm light, asdescribed above. Scale bar, 5 µm. (c) A pseudo-colored FRET image (right)shows the ratio of donor (CFP) emissions before (left) and after (central) YFPphotobleaching. Scale bar, 5 µm. The pseudo color scale marks the FRETratio from 0 (blue) to 6.0 (red). Results are typical of at least threeindependent experiments. Comparison of the FRET ratios for the interactionbetween CFP-CHFR and PML-YFP in living and fixed cells (n = 10 for each)shows three-fold lower FRET in living cells (data not shown).

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Notably, small CHFR foci occur not only in the nucleoplasm, butalso in the form of appendages on the surface of PML bodies.Comparison of the FRET images in Figures 2 and 3 suggests thatCHFR and PML do not interact in the paired appendages. On average,4.2 ± 1.3 pairs of GFP-CHFR foci decorate any given PML body,accounting for ∼ 20% of all small CHFR foci.

A dynamic interplay between GFP-CHFR appendages and PML bod-ies is apparent in time-lapse images of GFP-CHFR (Fig. 4b). Small GFP-CHFR-containing foci approach and fuse with PML bodies (Fig. 4b,0–135 s). Conversely, paired GFP-CHFR-containing foci can apparentlybe extruded from PML bodies over time (Fig. 4b, 190–250 s). Similarfusion and extrusion events are observed when serial, 1 µm z-planeimages through entire cells at each time point are projected into a singleplane (Supplementary Video 1 online), suggesting that the events trulyoccur at the surface of PML bodies, and are not simply the result ofchanges in the plane of focus of the foci. Collectively, these observationssuggest that deposition and loading of CHFR from or into the smallerfoci takes place on the surface of PML bodies, consistent with the alteredinteraction between CHFR and PML at the periphery of PML bodiesrevealed by FRET analysis (Fig. 2d).

Behavior of a trans-dominant inhibitory CHFR mutantA CHFR deletion mutant (CHFR∆FHA) lacking the N-terminalFHA domain (Fig. 1a) acts as a trans-dominant inhibitor of endoge-nous CHFR function9. When expressed in CHFR-proficient cells,this mutant abrogates the transient, CHFR-dependent delay in entryinto mitosis triggered by microtubule poisons such as nocodazole. Toexplore the correlation between the localization of CHFR and itsfunction, we investigated the effects of this trans-dominant mutanton GFP-CHFR distribution.

Myc epitope-tagged CHFR∆FHA was coexpressed in HeLa cellswith the GFP-CHFR fusion protein. Coexpression prevents the typicalfocal accumulation of GFP-CHFR in interphase cells (Fig. 5a). Neitherlarge nor small foci occurred. Instead, both proteins assumed a diffusenuclear distribution. Thus, inhibition of CHFR function by the

CHFR∆FHA mutant is accompanied by the loss of localization innuclear foci.

However, the CHFR∆FHA mutant had no effect on the distributionof PML (Fig. 5b). In cells coexpressing PML-GFP with the myc-taggedCHFR∆FHA mutant, PML-GFP continued to accumulate in character-istic PML bodies. This implies that the trans-dominant CHFR∆FHAmutant works to prevent the localization of CHFR to PML bodies andnot vice versa. Corroborating this conclusion, CHFR∆FHA did notcoimmunoprecipitate with PML or SUMO-modified forms of PML(data not shown). Collectively, our findings suggest that CHFR recruit-ment to PML bodies requires its FHA domain, a known phosphopep-tide-binding motif, and—because the CHFR∆FHA mutant suppressesCHFR function as a dominant-negative9—that this recruitment is rele-vant to biological function.

Control of CHFR dynamics by PMLTo investigate how the PML-CHFR interaction might regulate func-tion, we used fluorescence recovery after photobleaching (FRAP) todetermine the dynamics of movement of GFP-CHFR and the GFP-CHFR∆FHA mutant between PML bodies and the nucleoplasm (Fig. 6). Cumulative results of 30 experiments are tabulated (Table 1).Fluorescence recovery of GFP-CHFR in the nucleoplasm exceeds 90%of the prebleach intensity, indicating that the molecule is highlymobile. This behavior is shared with the GFP-CHFR∆FHA mutant,which localizes exclusively to the nucleoplasm. In contrast, GFP-CHFR localized to PML bodies has restricted mobility. Over the 20-stime frame of our measurements, fluorescence recovery is no morethan ∼ 50% of the prebleach intensity, indicating that at least half of theGFP-CHFR in PML bodies is in an immobile pool. The retardation of

Figure 4 Movement of small GFP-CHFR-containing foci to and from PMLbodies. (a) Cells expressing GFP-CHFR (green) were fixed and stained withanti-PML (red). Each image shows a single composite projection renderedfrom ∼ 60 individual z-planes, and highlights a single PML body. Regions ofcolocalization appear yellow in the merged images. GFP-CHFR-containingappendages, devoid of PML, occur on the surface of PML bodies. Scale bar,1 µm. (b) Time-lapse images show that small GFP-CHFR-containing fociapparently approach and coalesce with PML bodies (red circles), and alsoappear to be extruded from them (blue circles). Composite projections fromserial z-plane images through the entire nucleus at each time point indicatethat most CHFR-containing foci do not migrate out of the plane of focus(Supplementary Video 1 online). Results are typical of at least threeindependent experiments.

Figure 5 CHFR∆FHA prevents recruitment of CHFR, but not of PML, intoPML bodies. (a,b) Cells cotransfected with Myc-epitope-tagged CHFR∆FHA,and either GFP-CHFR (a) or PML-GFP (b) were fixed and stained with anti-Myc. Myc-CHFR∆FHA expression prevents GFP-CHFR from localizing to PMLbodies (a), but has no effect on the accumulation of PML-GFP in thesestructures (b). Scale bar, 5 µm.

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GFP-CHFR mobility in PML bodies is further substantiated by thereduced coefficient of diffusion. Thus, our results clearly demonstratethat the PML-CHFR interaction reduces the mobility of—or, in otherwords, sequesters—GFP-CHFR in PML bodies.

The dynamic behavior of GFP-CHFR sequestered in PML bodiesis not identical to that of PML or SUMO-modified PML in thesestructures. The fluorescence recovery of YFP-SUMO1 or of PML-GFP fusion proteins in PML bodies (14% or 4%, respectively) is farlower than that of GFP-CHFR (∼ 50%) there (Table 1). This is truewhether just half of a PML body is photobleached, or whether anentire one is (data not shown), indicating that PML-GFP in PMLbodies does not undergo exchange either with the nucleoplasm, orwithin the structure itself. We infer from these findings that PML orSUMO conjugates within PML bodies are immobile, whereas ∼ 50%of CHFR in PML bodies is mobile and can undergo exchange withother compartments. By distinguishing the dynamics of structuralcomponents of PML bodies from that of a protein that trafficsthrough them, our findings provide new insight into the roleascribed to PML bodies as repositories for protein deposition andmobilization during biological processes.

Distribution and dynamics of CHFR in PML–/– cellsThe trans-dominant inhibitory mutant, CHFR∆FHA, prevents theinteraction of CHFR with PML but does not perturb PML bodyintegrity (Fig. 5), suggesting that the traffic of CHFR through PMLbodies is necessary for function. This prompted us to examine thedistribution and dynamics of CHFR in primary cultures of PML–/–

murine embryo fibroblasts (MEFs), in which PML bodies are absent4.MEFs from litter mate, PML+/+ embryos were used as controls.

In PML–/– cells, GFP-CHFR did not accumulate in foci in thenucleus of interphase cells (Fig. 7a), but instead was diffusely distrib-uted in the nucleoplasm. Analysis by FRAP reveals that nucleoplasmic

GFP-CHFR in PML–/– cells recovers to >90% of its prebleach intensity(data not shown), suggesting that it resides in a freely mobile pool. Thedistribution and dynamics of GFP-CHFR in PML–/– cells closelyresemble those of the GFP-CHFR∆FHA mutant, which does not inter-act with PML, in both PML–/– and control cells. These findings con-firm that the PML-CHFR interaction in PML bodies is required for thecontrol of CHFR distribution and dynamics.

Mitotic abnormalities in PML–/– cellsCHFR-deficient cells, or cells transfected with the CHFR∆FHAmutant that does not interact with PML, do not exhibit the transientdelay in mitotic entry that occurs in wild-type cells after challengewith agents that disrupt the mitotic spindle9. So, we tested whetherPML bodies are also required for this response. The mitotic distribu-tion of asynchronously growing PML–/– and control cells was firstdetermined (Fig. 7b). Cells with condensed chromosomes but anintact nuclear envelope characteristic of the late G2–prophase transi-tion were over-represented in PML–/– cells as compared with wild-type controls, suggesting that prophase entry, duration or exit mightbe affected by PML disruption. Indeed, the CHFR-dependent check-point response is known to regulate this phase, delaying entry intomitosis after exposure of cells to microtubule poisons9. To test theeffect of PML disruption on prophase entry more directly, we usedpreviously described methods14. In contrast to control cells, whichprogressively accumulate in late G2–prophase with an intact nuclearmembrane after exposure to depolymerizing concentrations of noco-dazole, PML–/– MEFs continue to enter mitosis and accumulate inprometaphase, consistent with spindle assembly checkpoint arrest(Fig. 7c). Together, these observations indicate that PML is requiredto enforce the early, CHFR-dependent checkpoint that delays entryinto mitosis after spindle disruption, but not for the laterprometaphase arrest that is independent of CHFR.

DISCUSSIONWe report here that the intranuclear distribution and dynamics ofCHFR are controlled by its interaction with PML, identifying a newmechanism in the mitotic checkpoint response to microtubule poisons,and providing insight into the nature and function of PML bodies. Weused a GFP-CHFR fusion to track the intracellular distribution ofCHFR. Antibodies for fluorescent staining of the endogenous human

Table 1 CHFR dynamics by FRAP analysis

Mobile Diffusion

fraction (%) coefficient (µm s–1)

GFP-CHFR∆FHA 95.9 ± 4.2 1.7 ± 0.18

GFP-CHFR (nucleoplasm) 90.8 ± 6.8 0.83 ± 0.2

GFP-CHFR (PML body) 51.5 ± 6.5 0.21 ± 0.09

PML-GFP (PML body) 14.3 ± 1.8 0.14 ± 0.02

YFP-SUMO1 (PML body) 4.1 ± 2.1 0.04 ± 0.02

Maximum fluorescence recovery values (percent of prebleach intensity ± s.d.)calculated from the experimental recovery curves are shown, as are the effectivediffusion coefficients. Results are typical of three independent experiments, eachinvolving ten FRAP determinations. A typical experiment is shown in Figure 6.

Figure 6 Control of CHFR dynamics in PML bodies. FRAP analysis of livingHeLa cells transfected with the indicated constructs. Fluorescence intensitywas measured in the regions ringed in white before (prebleach) or atdifferent times after photobleaching to calculate flourescence recovery.Scale bar, 5µm.

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protein have not been reported, and our own attempts to raise rabbitanti-human CHFR antisera have not so far yielded a reagent that worksfor fluorescent staining. However, several lines of evidence indicate thatGFP-CHFR reliably reports the behavior of the endogenous protein.Antibody staining of the X. laevis ortholog of CHFR in X. laevis cellsreveals accumulation in large and small foci closely similar to GFP-CHFR distribution in human cells, as well as colocalization of endoge-nous CHFR with transfected GFP-CHFR. In transfected human cells,this focal distribution is not observed with other GFP fusion proteins, orwith GFP alone. Deletion of the FHA domain of CHFR is sufficient toprevent focal accumulation. Moreover, overexpression of theCHFR∆FHA mutant blocks the transport of coexpressed GFP-CHFR tofoci in a trans-dominant fashion, consistent with the known behavior ofthis mutant as a functional ‘dominant negative.’ Collectively, these find-ings suggest that the focal distribution of GFP-CHFR is relevant to thebiological properties and function of the endogenous protein.

Large CHFR-containing foci are coincident with PML bodies, and theinteraction between these proteins occurs only in these structures,involving a distinct pool of CHFR. Intermolecular FRET between CFP-CHFR and PML-YFP occurs exclusively in PML bodies, and not in smallnuclear foci that contain CHFR, or in the nucleoplasmic pool. FRAPexperiments indicate that ∼ 50% of CHFR in PML bodies undergoesexchange with other cellular pools. In fact, small CHFR-containing focimove to and from PML bodies. These observations suggest that intracel-lular CHFR cycles through PML bodies, with a substantial fraction resi-dent in these structures at any given time.

Traffic through PML bodies is essential for CHFR’s biological func-tion. Perturbation of the CHFR-PML interaction by the trans-dominantCHFR∆FHA mutant, or in PML–/– cells, not only alters the distributionand dynamics of CHFR, but is also accompanied by an inability toenforce the CHFR-dependent checkpoint response to microtubule dis-ruption. Protein activation in PML bodies putatively occurs via post-translational modifications such as acetylation, phosphorylation orSUMO conjugation8. Indeed, CHFR undergoes phosphorylation inmitotic cells15. We have not yet been able to detect such modificationson Myc-epitope-tagged CHFR or a GFP-CHFR fusion protein duringinterphase (data not shown), either because of the limitations of our

experimental system, or because CHFR is not modified during its tran-sit through PML bodies.

Recruitment of CHFR to PML bodies requires its N-terminal FHAdomain, a phosphopeptide-binding module that recognizes motifs sur-rounding modified serine or threonine residues. Our findings suggestthat phosphorylated PML may itself be the anchor for CHFR recruit-ment, because intermolecular FRET shows the molecules to be in closephysical proximity in PML bodies. Indeed, PML can be phosphorylatedon tyrosine and serine residues16. Notably, the trans-dominantCHFR∆FHA mutant not only fails to interact with PML but also pre-vents the recruitment of GFP-CHFR to PML bodies, indicating the for-mation of a CHFR–CHFR∆FHA heterodimer. Dimerization may reflectthe fact that CHFR is active as an E3 ubiquitin ligase through its RINGdomain10,15,17, because these enzymes usually comprise hetero- orhomodimers of RING-containing proteins18. PML itself contains aRING domain1, prompting speculation that the PML-CHFR interac-tion might have enzymatic activity.

Notably, FRET analysis reveals heterogeneity in the interactionbetween CHFR and PML within PML bodies. A central core regionwith a high FRET ratio is bounded by a thin peripheral rim in whichthe FRET interaction is diminished. Indeed, the movement of smallCHFR-containing foci to and from PML bodies involves the peripheralrim, suggesting that the PML-CHFR interaction might be structurallyor quantitatively altered in this region. Whether these differences arereflected in visible ultrastructural features of the organization of PMLbodies into a dense core with a central aperture19 is not known, but ourfindings suggest that this warrants further study.

We find using FRAP analysis that two structural components inPML bodies (PML itself, and PML body–bound SUMO conjugates)are comparatively immobile, in that only ∼ 5–15% of these moleculescan be exchanged with surrounding compartments. In contrast, atleast one dynamic component, CHFR, is relatively more mobile. Ourobservations provide insights into the role of PML bodies as sites forprotein storage or modification. They suggest that dynamic and struc-tural components of PML bodies are neither stoichiometrically norpermanently associated with one another, but instead are in a dynamicequilibrium. This feature could facilitate the movement of proteins

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Figure 7 Perturbed CHFR localization and mitotic checkpoint abnormalities after PML disruption. (a) PML+/+ or PML–/– MEFs transfected with either GFP-CHFR∆FHA or GFP-CHFR were fixed and visualized. CHFR focus formation is impaired in PML–/– MEFs. Scale bar, 5 µm. (b) Enumeration of mitotic stages(G2–Pro, cells in late G2 phase or prophase, with intact nuclear membranes; Prometa, prometaphase; Meta, metaphase; Ana, anaphase; Telo, telophase) inasynchronously dividing PML+/+ or PML–/– MEFs. The frequency of cells in late G2–prophase is elevated in PML–/– cells. Over 4,000 cells were enumerated ineach of two independent experiments. (c) Cells in different mitotic stages were enumerated before (0), and at 90 and 270 min after, treatment with 10 µMnocodazole as described14. Cells in metaphase, anaphase or telophase were grouped together as ‘late mitosis’ for convenience. In contrast to PML+/+ cells,which show an arrest in late G2–prophase, PML–/– cells breach the CHFR-dependent checkpoint to accumulate in prometaphase. Their accumulation inprometaphase indicates that the spindle assembly checkpoint triggered by nocodazole, not dependent on CHFR, is unaffected by PML disruption. Over4,000 cells were enumerated in each of two independent experiments.

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1120 VOLUME 11 NUMBER 11 NOVEMBER 2004 NATURE STRUCTURAL & MOLECULAR BIOLOGY

like CHFR to and from PML bodies. In this light, the relative immobil-ity of PML and SUMO conjugates in the PML body is notable, giventhat they are believed to work directly in protein storage or modifica-tion. Our findings suggest that the regulated mobilization of PMLand/or SUMO by cellular signals might be essential to induce proteinrelease or modification.

The intranuclear distribution and dynamic properties of GFP-CHFR are altered in PML–/– cells, providing an independent line ofevidence to support our observations on the CHFR∆FHA mutant.Notably, cell cycle progression in PML–/– cells is subtly perturbed, inthat late G2–prophase cells are more abundant, suggesting alterationsin mitotic entry or prophase progression. This is not a reported featureof CHFR deficiency9, raising the possibility that it could reflect a func-tion of PML that is independent of CHFR.

CHFR is the only known component of the checkpoint mecha-nism that delays entry into mitosis in response to microtubule poi-sons, an important and widely used class of anti-cancer drugs. Thecheckpoint is frequently inactivated in human epithelial cancers, bymutations or epigenetic silencing of CHFR20–23. We report here thatPML–/– cells fail to exhibit the delay in mitotic entry after micro-tubule disruption that characterizes this CHFR-dependent check-point, indicating that PML also participates in the mechanism.Moreover, the CHFR∆FHA mutant — which suppresses the check-point in a trans-dominant manner — not only fails to accumulate inPML bodies, but also prevents CHFR from doing so. Thus, multiplelines of evidence indicate that the PML-CHFR interaction is essen-tial for this checkpoint. It will therefore be important to ascertainwhether alterations in the expression of PML, or mutations whichaffect its ability to control CHFR, also compromise the checkpointin human cancer cells, influencing their sensitivity to treatmentwith microtubule-disrupting drugs. This is particularly pertinent inlight of the observation that PML is frequently inactivated in humanepithelial cancers from diverse tissues5.

METHODScDNA cloning. cDNAs encoding full-length CHFR and the CHFR∆FHAmutant were isolated by S. Anand (Hutchison/MRC Center) through RT-PCRreactions on RNA prepared from 293T cells using RNAzol. The oligonu-cleotide primers used for RT-PCR incorporated EcoRI (5′) and XhoI (3′)restriction sites for cloning into the vector pCS2MT, thereby adding 6x in-frame Myc epitope tags to the 5′ end. The cDNAs (without Myc tags) were sub-cloned as EcoRI (5′) to XbaI (3′) fragments into pEGFP-C1 (Clontech). Theprimers used were (Forward) 5′-ACGCGTGAATTCCATGGAGCGGCCC-GAGGAAG-3′ and (Reverse), 5′-TGCAAGATCTCTCGAGTTAGTTTTT-GAACCTTGTCTGTTCACAGATA-3′, for full-length CHFR. The sameReverse primer was paired with (Forward) 5′-ACGCGTGAATTCCATG-GAACCGGAACACAACGTGGCATACCTC-3′ for CHFR∆FHA. CFP and YFPwere subcloned in place of EGFP between NheI and EcoRI sites. PML-YFP-N1was a gift from T. Rich. YFP was replaced with GFP from pEGFP-N1(Clontech) via BamHI and NotI restriction sites. Constructs were verified bynucleotide sequencing.

Xenopus laevis CHFR isolation and analysis. To clone a cDNA encoding x-CHFR, the National Center for Biotechnology Information database ofX. laevis ESTs was probed with the protein sequence of human CHFR using thetranslated BLAST algorithm. RT-PCR was used to assemble a full-length clonefrom X. laevis RNA prepared from embryos, supplemented by the RACEmethod to complete the 5′ end. The sequence and properties of the protein willbe reported separately. A rabbit antiserum was raised against residues 277–420expressed as a glutathione S-transferase fusion protein, and affinity-purifiedagainst antigen. The affinity-purified antibody was validated in western blot-ting against oocyte extract, using as a positive control in vitro translated proteinprepared using the TnT reticulocyte lysate system (ProMega) from full-lengthx-CHFR cDNA. XR1 cells24, a gift from W.A. Harris (University of Cambridge),

were transfected with the GFP-CHFR construct by DNA microinjection forcolocalization experiments. Antibody staining and microscopic imaging weredone according to the methods below.

Transfection. Cell lines or primary MEFs were transfected withLipofectamine 2000 (Invitrogen) according to the manufacturer’s instruc-tions and analyzed by western blot, immunofluorescence or live cellmicroscopy at 36 h after transfection.

Antibodies. Anti-PML mouse monoclonal (PG-M3) antibody was purchasedfrom Santa Cruz Biotechnology and used at 1:200 for western blot, immunopre-cipitation and immunofluorescence. Purified anti-GFP polyclonal (ab290-50,Abcam) antiserum was used for immunoprecipitation, and anti-GFP mono-clonal (8362-1, Clontech) for western blot. Monoclonal anti-Myc (9E10) waspurchased from Santa Cruz Biotechnology and used at 1:1,000 for western blotand immunofluorescence. Fab fragment secondary antibodies for immunofluo-rescence were purchased from Molecular Probes and used at 1:200.

Immunofluorescence. Cells grown overnight on 0-thickness glass coverslips(VWR) were fixed for 5 min at room temperature in 4% (w/v) formaldehyde,washed with TBS containing 0.1% (v/v) Triton-X100 and blocked in AbDil (2%(w/v) BSA, 0.1% (w/v) sodium azide in TBS with 0.1% (v/v) Triton X-100).AbDil was used to dilute primary and secondary antibodies, before incubationat room temperature for 3 h or 1 h, respectively. Slides were mounted inVectashield with or without DAPI.

Immunoprecipitation. Transfected cells (3 × 106) were lysed in 150 mM NaCl,0.1% (v/v) NP40, 100 µM sodium orthovanadate, 100 µM PMSF, 1 mM DTT,on ice. Cell debris was pelleted by centrifugation before the cleared supernatantwas aliquoted for immunoprecipitation with 2 µg of anti-PML, anti-GFP poly-clonal or control rabbit IgG. Antibody capture was done with proteinG–Sepharose beads (Sigma), which were washed extensively in lysis bufferbefore elution of immunoprecipitates into SDS-PAGE loading buffer.

Microscopy. All images were acquired on a Zeiss Axiovert 200M invertedmicroscope equipped with a Zeiss 510 META confocal head, using Zeiss soft-ware. Three-color confocal images were obtained by laser excitation usingappropriate band-pass filters. For live cell imaging, a heated stage unit (HeatingInsert P, Zeiss) was used to incubate 1-thickness borosilicate chambered cover-glass slides (Nunc) layered with cells growing in Liebowitz L-15 medium with-out phenol red (Invitrogen) supplemented with 10% (v/v) fetal calf serum.Two-pixel images showing quantitative colocalization of X-CHFR with GFP-CHFR were generated using a standard function in the Zeiss LSM510 software.Briefly, a bivariate scatter plot was produced by the software, displaying fluores-cence intensities for each channel for each image pixel, before threshold deter-mination and quantitative colocalization analysis were done as described25 togenerate the colocalization image.

FRET. FRET analysis by acceptor photobleaching was carried out as described26,with slight modifications. Briefly, HeLa cells transfected with either CFP-CHFRor YFP-PML alone were plated onto coverslips and emission spectra obtained(data not shown). Cells doubly transfected with both fusion proteins were usedfor acceptor photobleaching hetero-FRET experiments. Lambda-mode excita-tion using 405-nm light from a blue diode laser at 9.9% laser power was used toextract spectral emissions for CFP and YFP. FRET efficiency was estimated(reviewed in ref. 13) for each image according to EFRET = ICFPpost / ICFPpre.

FRAP. Cells were visualized with a 40× 1.3-NA oil objective lens with 10× digi-tal zoom, imaged at 256 × 256 8-bit resolution with a scan speed of 245 msusing 488-nm argon laser light at 1% power. A 2-µm bleach spot was generatedafter 5 scans, using 30 iterations of 100% power 488-nm light, before imageacquisition to monitor fluorescence recovery. Data were corrected for the lossof fluorescence owing to imaging as described27. Each data point represents theaverage of 30 observations acquired from 3 independent experiments. Theeffective diffusion coefficient was estimated from the solution of D = βα2 / t,where β accounts for the relative mobility of each component as described28.

Mitotic progression and arrest. MEFs from PML–/– embryos or PML+/+ littermate controls4 in passage 3 of culture were plated at 500,000 cells per well in a

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six-well plate containing a 0-thickness glass coverslip in DMEM supplementedwith 10% (v/v) FCS. After 16 h, the coverslips were fixed in 0.5% (v/v) glu-taraldehyde for 7 min at room temperature, and quenched in 0.2% (w/v)sodium borohydride. The coverslips were washed extensively with TBS 0.1%(v/v) Triton-X-100 and mounted in Vectashield plus DAPI (Vecta Labs). Theclassic mitotic phases29 were scored on the basis of chromosome morphology,with >4,000 cells counted per sample. To assess the mitotic response to spindledisruption, cells were treated with a final concentration of 10 µM nocodazole(Sigma) added from a 1.7 mM stock for the indicated times before processing asabove, as described14.

Note: Supplementary information is available on the Nature Structural & MolecularBiology website.

ACKNOWLEDGMENTSM. Daniels generously assisted us with XR1 cell culture and preparation of X. laevisextracts, 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 theMB/PhD program at the University of Cambridge, and by the UK MedicalResearch 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 andCancer Research UK.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 15 July; accepted 3 September 2004Published online at http://www.nature.com/nsmb/

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