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Mobile foci of Sp100 do not contain PML: PML bodies are immobile but PML and Sp100 proteins are not
Journal of
Structural Biology Journal of Structural Biology 140 (2002) 180–188 www.academicpress.com
Mobile foci of Sp100 do not contain PML: PML bodies are immobile but PML and Sp100 proteins are not Karien Wiesmeijer, Chris Molenaar, Ivory M.L.A. Bekeer, Hans J. Tanke, and Roeland W. Dirks* Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, Leiden 2333 AL, The Netherlands Received 1 July 2002, and in revised form 12 September 2002
Abstract PML bodies are nuclear organelles that are associated with various diseases and are suggested to be involved in multiple cellular activities including transcriptional regulation, apoptosis, and antiviral defence. Because many proteins with different functions aggregate in PML bodies, it has also been suggested that these bodies function as nuclear depots. Some proteins consistently found in PML bodies may form a stable scaffold that regulates the recruitment of other proteins. Thus, some proteins might be stably integrated into PML bodies while others continuously exchange with the nucleoplasm. To study the dynamic properties of PML bodies and resident proteins, we constructed fusion proteins of Sp100, PML, and CBP with autofluorescent proteins. Using timelapse imaging, we show that PML bodies exhibit little movement but that small foci that contain Sp100 but not PML are dynamic and fuse with PML bodies. Furthermore, we show by monitoring fluorescence recovery after photobleaching that Sp100, PML, and CBP are dynamic components of PML bodies. This suggests that these proteins do not play a strict structural role in these bodies but that they function at other sites in the nucleoplasm. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: PML; Sp100; CBP; Time-lapse imaging; FRAP
1. Introduction Our view of how the cell nucleus is spatially and functionally organized has changed dramatically since it has become possible to study the localization and kinetics of nuclear components in living cells. With the advent of probe and green fluorescent protein (GFP) technologies as well as live cell imaging methods, chromatin dynamics, RNA transport, and protein localization and kinetics have been extensively studied. These studies have revealed that chromatin can be highly dynamic (Gasser, 2002; Tumbar et al., 1999), that RNAs are transported throughout the interchromatin space (Dirks et al., 1999, 2001; Politz et al., 1999), and that proteins involved in diverse nuclear processes are highly mobile (Chen et al., 2002; Dundr and Misteli, 2001; Houtsmuller et al., 1999; Leung and Lamond, 2002; *
Corresponding author. Fax: +31-0-71-5276180. E-mail address: [email protected] (R.W. Dirks).
Misteli et al., 1997; Snaar et al., 2000). Many nuclear proteins reside in nuclear compartments and, by tagging these proteins with GFP, the dynamic behavior of these compartments could be analyzed by time-lapse microscopy. Cajal bodies, for example, were shown to move in the nucleoplasm and occasionally were shown to fuse to form larger bodies and to split from larger bodies (Platani et al., 2000; Snaar et al., 2000). The functions of most nuclear compartments have not been completely resolved yet and this is particularly true for PML bodies. PML bodies, also referred to as ND10 (nuclear domain 10) or PODs (PML oncogenic domains), range in size from 0.2 to 1 lm and are present in 10–30 copies per nucleus. They fluctuate in size and number during the cell cycle (Everett et al., 1999). Since the identification of Sp100 as a component of PML bodies (Szostecki et al., 1990), many other proteins have been found present in these compartments (Matera, 1999; Negorev and Maul, 2001). Because these proteins fulfill multiple biological functions, PML
1047-8477/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 7 - 8 4 7 7 ( 0 2 ) 0 0 5 2 9 - 4
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bodies have been implicated to play a role in various cellular processes including the control of chromatin or heterochromatin architecture (Seeler et al., 1998), regulation of apoptosis (Guo et al., 2000; Wang et al., 1998), transcription (LaMorte et al., 1998), MHC class I antigen presentation (Zheng et al., 1998), detection of foreign protein or protein–nucleic acid complexes and proteolytic degradation (Everett et al., 1997; Tsukamoto et al., 2000), antiviral defense (Ishov and Maul, 1996), and cell proliferation and tumor suppression (Salomoni and Pandolfi, 2002). It cannot be excluded, however, that the main or only function of PML bodies may be to regulate the recruitment and release of proteins in order to control their availability at nucleoplasmic sites other than PML bodies as suggested by Negorev and Maul (2001). In their view, PML bodies function as a nuclear depot (Maul, 1998). Essential for the assembly and stability of PML bodies are PML and its modification by the small ubiquitin-related protein SUMO-1 (M€ uller et al., 1998). Cells lacking PML or expressing PML– RARa and RARa–PML fusion proteins, as in acute promyelocytic leukemia cells, do not reveal PML bodies. Less well understood is the role of SUMO-1 modification in PML body assembly because studies using PML mutants that cannot be SUMO-modified provide contradictory results (Ishov et al., 1999; M€ uller et al., 1998). Therefore, SUMO modification has been proposed to be a control mechanism in the accumulation of proteins at PML bodies. For example, SUMO modification of PML proved essential for the recruitment of Daxx, a protein enriched in condensed chromatin, to PML bodies (Ishov et al., 1999). Also, other proteins, including CREB binding protein (CBP), Blooms (BLMs), and Sp100, do not localize at PML bodies in the absence of PML, though SUMO modification of PML does not seem to play a role in their recruitment. In contrast to PML, SUMO-1 modification of Sp100 does not appear to play a role in its deposition at PML bodies (Sternsdorf et al., 1997). How proteins are recruited to PML bodies is still enigmatic but adaptor proteins and protein–protein interactions are assumed to be essential to this process. On the basis of their kinetic behavior, PML and Sp100 are suggested to play structural roles in PML bodies (Boisvert et al., 2001). Fluorescence recovery after photobleaching (FRAP) analysis of GFP–PML and GFP–Sp100 movement revealed that these chimeric proteins are immobile inside PML bodies (Boisvert et al., 2001). At the same time, another PML inhabitant, the transcription coactivator CBP, was shown to move rapidly into and out of these bodies. The observation that PML and Sp100 are immobile in PML bodies is in contradiction to the hypothesis that nuclear compartments are the reflection of steady-state association/dissociation of its residents with the nucleoplasm (Phair and Misteli, 2000). In fact, proteins that localize at di-
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verse nuclear compartments were shown to exhibit rapid movements (Kruhlak et al., 2000; Phair and Misteli, 2000; Snaar et al., 2000). We therefore reevaluate the kinetic behavior of PML, Sp100, and CBP and show that all three proteins are dynamic components of PML bodies. In addition, using time-lapse fluorescence microscopy, we show that PML bodies exhibit little movement in interphase cell nuclei but that Sp100 foci that do not contain PML exhibit more extended movement. The generally small-sized Sp100-containing foci appear to fuse with larger PML bodies but not to separate from them. These observations suggest that homooligomerization of Sp100 in the nucleoplasm can occur in the absence of PML and precede recruitment to PML bodies.
2. Materials and methods 2.1. Cell culture VH10 human primary fibroblasts and U2OS human osteosarcoma cells were cultured on 3.5-cm glass bottom petri dishes (Mattek, Ashland, MA) in DMEM without phenol red containing 1.0 mg/ml glucose, 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 lg/ ml streptomycin and buffered with 25 mM Hepes buffer to pH 7.2 (all from Invitrogen). Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. 2.2. Plasmid constructs and cell transfection Full-length PML in an EGFP–C1 vector was a gift from Dr. A.G. Jochemsen (LUMC, Leiden). The EGFP fragment was replaced by ECFP and EYFP using the restriction sites AgeI and BsrGI. Sp100 cDNAs were amplified from total RNA isolated from U2OS cells using reverse transcription PCR and the forward primer 50 GCGCGCGGTACCATGGCAGGTGGGGGCCAG-30 containing the KpnI site and the reverse primer 50 GCGCGCCCCCGGCTAATCTTCTTTACCTGACCC-30 containing the XmaI site. The cDNA for CBP was PCRamplified from a plasmid containing full-length CBP (provided by Dr. E. Kalkhoven, LUMC, Leiden). Purified amplified PCR fragments were inserted in-frame into the KpnI–XmaI fragment of pEYFP–C1 and pECFP–C1 (Clontech Laboratories) and verified by sequencing. Transient transfections of cells were performed at approximately 60–80% confluence using DOTAP under conditions recommended by the manufacturer (Roche Diagnostics GmbH). 2.3. Immunofluorescence Cells grown on microscopic glass slides were washed in PBS and fixed with 4% formaldehyde in PBS for 5 min
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at room temperature. Subsequently, cells were permeabilized in PBS containing 1% Triton X-100 for 15 min and washed in Tris-buffered saline (TBS), pH 7.4. Cells were then incubated for 45 min at room temperature with the mouse anti-PML antibody 5E10 (Stuurman et al., 1992) diluted 1:10 in TBS containing 0.5% (w/v) blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany). Following three rinses in TBS containing 0.05% Tween, cells were incubated with the secondary antibody goat anti-mouse Alexa 594 (Molecular Probes, Eugene, OR, USA; dilution 1:1000) for 45 min, washed in TBS, and mounted in Vectashield (Vector Laboratories, Burlingame, California) containing 50 lg/ml 40 ,60 -diamidino-2-phenyl indole (DAPI). 2.4. Time-lapse microscopy Approximately 16 h after transfection, three-dimensional time-lapse images were recorded on a Leica TCS/ SP2 confocal microscope system. During imaging, the cells were kept in a closed chamber and the temperature of the culture medium was kept at 37 °C using a temperature-controlled ring (Harvard Apparatus), which was placed around the culture chamber, and a microscope objective heater (Bioptechs, Butler, PA). Image stacks of cells were acquired using a 100 NA 1.4PL APO lens and analyzed with Leica software. For each nucleus, 5–10 optical sections separated by 0.5 lm were acquired every 1–5 min over a period of 1–2 h. In total for each transfection experiment 10 nuclei were recorded. Images were viewed as maximum intensity projections of each time point. 2.5. Frap and flip FRAP and FLIP were performed on the Leica TCS/ SP2 confocal microscope system using spot bleaching. For FRAP experiments, 5–6 confocal sections were acquired followed by a single bleach pulse of 6 s in FRAP and multiple bleach pulses of 0.5–6 s/body or region in FLIP. Then, images of multiple sections were collected at time intervals of 20–60 s. Quantitative analysis of the amount of fluorescence in bleached regions relative to unbleached regions was performed with the software options delivered with the Leica system and with Excel. Corrections were made for background and for fluorescence fading that occurred during the measurements.
3. Results and discussion 3.1. Fusion proteins of PML, Sp100, and CBP with EYFP or ECFP localize to PML bodies PML, Sp100, and CBP were fused at their amino terminus to EYFP and to ECFP and transiently trans-
fected in VH10 fibroblasts and in U2OS cells. Before time-lapse experiments were started, the distribution of these chimeric proteins at PML bodies was confirmed by immunocytochemistry. EYFP– and ECFP–Sp100 and EYFP– and ECFP–PML were shown to localize at PML bodies in both cell lines. EYFP– and ECFP–CBP also localized at PML bodies, but generally at lower intensities, and the amount varied among cells. In addition, the CBP fusion proteins were shown to localize throughout the nucleoplasm, excluding nucleoli. These results show, in agreement with previous studies (Boisvert et al., 2001), that the GFP fusion proteins localize in the same manner as the endogenous counterparts. However, close examination of image stacks of cells containing EYFP–Sp100 revealed small Sp100 aggregates or foci that were not stained with the anti-PML antibody. The presence of foci that contain Sp100 but not PML became even clearer from cells that were double-transfected with EYFP–Sp100 and ECFP–PML or with ECFP–Sp100 and EYFP–PML. As shown in Fig. 1, these double-transfected cells allowed us to distinguish between PML bodies containing both PML (cyan in Fig. 1A) and Sp100 (yellow in Fig. 1B) and bodies that contain Sp100 only (small yellow bodies in Figs. 1B and C). Sp100 foci that do not contain PML have previously been interpreted as precipitates of Sp100 that could not be accommodated by PML bodies when Sp100 is extensively over expressed (Negorev et al., 2001). In this context, it has been suggested that the amount of binding sites for Sp100 in PML bodies would be limited and that Sp100 would not self-aggregate in these domains. These observations have been made, however, in fixed cells and do not exclude the possibility that multimerization of Sp100 may precede uptake by PML bodies. Interestingly, the N-terminal region of Sp100 (amino acids 33–149), which is important for dimerization, is also required for PML body targeting (Sternsdorf et al., 1999). Thus, it is very possible that homodimerization of Sp100 might be required to generate a functional PML body-targeting signal. Thus far, however, there is no experimental evidence that Sp100 might form multimers (Sternsdorf et al., 1999), though interactions with other proteins have been described. Therefore, it would be interesting to investigate whether other proteins than PML are present in Sp100 foci. Because HP1 is found present in PML bodies and is shown to interact with Sp100 (Seeler et al., 1998), it is a good candidate. 3.2. EYFP–Sp100 bodies are dynamic and fuse with PML bodies To test the idea that Sp100 may form aggregates in the nucleoplasm that have then the ability to fuse with PML bodies, we thought to perform time-lapse experiments using VH10 fibroblast as well as U2OS cells
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Fig. 1. Double expression of ECFP–PML and EYFP–Sp100 in U2OS cell. ECFP–PML (A) and EYFP–Sp100 colocalize in PML bodies (pink in C). A number of small Sp100-containing bodies can be distinguished that do not contain PML protein (yellow in C). Bar ¼ 10 lm.
transiently expressing EYFP–Sp100 to examine the dynamic behavior of Sp100-containing foci. For this purpose, cells containing low to moderate levels of EYFP–Sp100 have been visually selected on the basis of fluorescence intensity. The results revealed that the larger EYFP–Sp100 containing bodies, which were shown to correspond to PML bodies by immunocytochemistry, were relatively immobile while the smaller foci revealed significant movement. Notably, not all small-sized foci were dynamic during the time a cell was imaged. Mostly, two to five of the small-sized foci were shown to move during the time-span a cell was imaged (generally 2 h). Interestingly, the small EYFP–Sp100 foci were observed to fuse with larger bodies and occasionally with other small foci (Fig. 2). Muratani et al. (2002) recently reported the rapid movement of smaller PML bodies that were labelled with EYFP–Sp100. However, these bodies have been interpreted as PML bodies, while our
results show that these bodies do not necessarily contain PML and as such should not be named PML bodies. Our results suggest that Sp100 has the ability to form homomers in the nucleoplasm outside PML bodies and that the resulting aggregates or foci can fuse with PML bodies. At present, it is not known what drives the fusion of bodies or which interactions are involved to ‘‘store’’ Sp100 in PML bodies. To answer the last question, we are currently studying protein–protein interactions with possible candidates using fluorescence energy transfer (FRET) and fluorescence live cell imaging microscopy (FLIM). 3.3. Sp100, PML, and CBP are dynamic components of PML bodies Having shown that Sp100 foci can fuse with PML bodies, we addressed the question of whether Sp100
Fig. 2. Time-lapse images of VH10 cell expressing EYFP–Sp100. As indicated by arrows, small EYFP–Sp100 bodies move in the cell nucleus and fuse with larger PML bodies.
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would be a dynamic component of PML bodies. For this purpose, cells were transfected with EYFP–Sp100 and subjected to photobleaching. Because the level of expression of tagged Sp100 could affect the extent to which they move we selected cells expressing moderate levels of EYFP–Sp100. By using the spot bleach mode of the Leica confocal system, an optical section was scanned and 1–3 PML bodies containing EYFP–Sp100 were selectively marked. The intensity of the laser light was adjusted that in the selected areas, the fluorescence intensity was dropped by more than 90% following photobleaching during 6 s. Images were then collected each 20–60 s to follow the entry of unbleached EYFP– Sp100 into PML bodies. Recovery of fluorescence was already noticeable within 1 min after bleaching and nearly complete recovery of fluorescence was ob-
tained within 7 min (Fig. 3). This suggests that there is a continuous exchange of EYFP–Sp100 in PML bodies. We then repeated the FRAP procedure for cells expressing EYFP–PML or EYFP–CBP. EYFP–PML revealed kinetic behavior similar to that of EYFP–Sp100 (Fig. 4). A much more rapid exchange of EYFP–CBP was observed. Within 10 s after photobleaching, a full recovery of fluorescence of EYFP–CBP in PML bodies was observed, suggesting that CBP is highly dynamic in the cell nucleus. Also FLIP, experiments in which PML bodies or selected areas in the nucleoplasm outside PML bodies were bleached for extended periods revealed that CBP is highly dynamic. Already within 15 s, continuous photobleaching of a small marked area resulted in nearly complete loss of nuclear fluorescence (Fig. 5). Furthermore, fluorescence intensities within and outside
Fig. 3. FRAP of three PML bodies in a VH10 cell expressing EYFP–Sp100. The bleached areas are indicated by circles. Images are taken before (A), just after (B), and at regular time points after bleaching (C and D show examples). The fluorescence intensities in the bleached and in three unbleached areas were measured and expressed as relative intensities (after correction for background). The mean intensity values of the intensities measured in the bleached regions were calculated during the time-lapse experiment, corrected for fluorescence fading, and shown in a fluorescence recovery curve (E). This curve shows that full recovery of EYFP–Sp100 is obtained after 7–8 min.
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Fig. 4. FRAP of a PML body in a VH10 cell expressing EYFP–PML. The first image is taken before the marked PML body is bleached (A), the second just after it is bleached (B), and the third and fourth images are examples of time points collected after bleaching (C and D). The fluorescence intensity in the bleached region was measured each 20 s for 9 min, corrected for background fluorescence and for fluorescence fading, and expressed as relative intensities (E). The fluorescence recovery curve shows that full recovery of fluorescence is obtained after 8–9 min.
PML bodies were shown to decrease at the same rate. Also, FLIP experiments confirmed that PML and Sp100 are dynamic components of PML bodies although, as shown for EYFP–Sp100 (Fig. 6), it took significantly more time to bleach all PML and Sp100. These results are consistent with the view that nuclear proteins travel throughout the cell nucleus and transiently interact with other proteins, which may lead to their appearance in nuclear bodies (Phair and Misteli, 2000). Clearly, the time of interaction may vary among different proteins, as became apparent from the different residence times of CBP, Sp100, and PML in PML bodies. Our observation that CBP is highly dynamic is in agreement with results presented by Boisvert et al. (2001). However, our observations that Sp100 and PML are also dynamic components of PML bodies, although to a lesser extent than CBP, are in clear contradiction to
their observations. They observed very little recovery of GFP–PML and GFP–Sp100 even after 10 min, when half of the cell nucleus was photobleached. On basis of these recovery times, it was concluded that GFP–PML and GFP–Sp100 form relatively immobile complexes in the nucleoplasm as well as within PML bodies and play a structural role in PML bodies. This discrepancy in results cannot be easily explained but we followed a different approach to bleach PML bodies. Rather than bleaching half-nuclei, we selectively bleached PML bodies using spot bleaching. The advantage of this method is that movement of GFP fusion proteins that reside in the near vicinity of PML bodies is taken into account, and not only molecules that have to travel over large distances. Probably more important for the interpretation of our results is that we kept our cells at 37 °C during all
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Fig. 5. Determination of FLIP of EYFP–CBP that is expressed in a U2OS cell. The circle in the image taken before bleaching (A) indicates the area that is continuously photobleached while images were collected each second. The time series shows that after 15 s nearly all EYFP–CBP present in this cell is photobleached (B–H).
measurements, while Boisvert et al. performed their measurements at room temperature (22 °C), which slows down energy-dependent processes. Indeed, when we performed our FRAP measurements of EYFP–Sp100 and EYFP–PML at room temperature, we measured significantly longer recovery times. After photobleaching EYFP–Sp100 or EYFP–PML in a PML body, full
recovery of fluorescence was observed only after 20 min. This indicates that the incubation temperature of the cells has a strong impact on the mobility of Sp100 and PML and possibly of nuclear proteins in general. Furthermore, this temperature effect suggests that uptake of SP100 and PML by PML bodies requires energy or, alternatively, that these proteins are transported
Fig. 6. FLIP in a VH10 cell expressing EYFP–Sp100. Cells were imaged before bleaching (A) and at regular successive time points each preceded by a 6-s bleaching period of the indicated region in B. Nearly all EYFP–Sp100 fluorescence is lost within 10 min (B–H).
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through the nucleoplasm by an energy-dependent mechanism. Concerning the formation of PML bodies, we can only speculate how they are assembled. Apparently, PML is essential in this process, while other proteins may reside in these bodies because they interact directly or via other adaptor proteins with PML. As we have shown, Sp100 and PML are dynamic components of PML bodies. This makes sense because Sp100 as well as PML were reported to function at chromatin, which is not found present in PML bodies. For example, it has recently been shown that PML interacts with histone deacetylases (HDAC) and represses transcription by recruiting HDAC to target gene promoters (Wu et al., 2001). Like PML, Sp100 has been implicated as a factor that plays a role in transcriptional regulation. It has been shown to play a role in transcriptional repression by interacting with HP1 (Lehming et al., 1998; Seeler et al., 1998, 2001) and more recently to play a role in transcriptional activation by interaction with the transcription factor ETS-1 (Wasylyk et al., 2002). Interestingly, electron spectroscopic imaging analyses have revealed that some PML bodies are surrounded by blocks of chromatin (Boisvert et al., 2000). It is temptive to speculate that the transcriptional activity of the genes located in these domains is under the control of protein factors present in PML bodies, such as Sp100, PML, and CBP. This, however, does not preclude the possibility that Sp100 and PML also have specific functions within PML bodies. At least, for Sp100 and PML to be active in various biochemical pathways intact PML bodies are required (Salomoni and Pandolfi, 2002).
Acknowledgments We thank Alt Zantema, Eric Kalkhoven, and A.G. Jochemsen for PML and CBP plasmids. This work was supported by the Dutch Scientific Organization NWO program ‘‘4D Imaging of Living Cells and Tissues’’, Grant 901-34-144.
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nuclear matrix-associated nuclear bodies. J. Cell Sci. 101, 773– 787. Szostecki, C., Guldner, H.H., Netter, H.J., Will, H., 1990. Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis. J. Immunol. 145, 4338–4347. Tsukamoto, T., Hashiguchi, N., Janicki, S.M., Tumbar, T., Belmont, A.S., Spector, D.L., 2000. Visualization of gene activity in living cells. Nature Cell Biol. 2, 871–878. Tumbar, T., Sudlow, G., Belmont, A.S., 1999. Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain. J. Cell Biol. 145, 1341–1354. Wang, Z.-G., Ruggero, D., Ronchetti, S., Zhong, S., Gaboli, M., Rivi, R., Pandolfi, P.P., 1998. PML is essential for multiple apoptotic pathways. Nature Genet. 20, 266–272. Wasylyk, C., Schlumberger, S.E., Criqui-Filipe, P., Wasylyk, B., 2002. Sp100 interacts with ETS-1 and stimulates its transcriptional activity. Mol. Cell. Biol. 22, 2687–2702. Wu, W.-S, Vallian, S., Seto, E., Yang, W.-M., Edmondson, D., Roth, S., Chang, K.-S., 2001. The growth suppressor PML represses transcription by functionally and physically interacting with histone deacetylases. Mol. Cell. Biol. 21, 2259–2268. Zheng, P., Guo, Y., Niu, Q., Levy, D.E., Dyck, J.A., Lu, S., Sheiman, L.A., Liu, Y., 1998. Proto-oncogene PML controls genes devoted to MHC class I antigen presentation. Nature 396, 373–376.
Structural Biology Journal of Structural Biology 140 (2002) 180–188 www.academicpress.com
Mobile foci of Sp100 do not contain PML: PML bodies are immobile but PML and Sp100 proteins are not Karien Wiesmeijer, Chris Molenaar, Ivory M.L.A. Bekeer, Hans J. Tanke, and Roeland W. Dirks* Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, Leiden 2333 AL, The Netherlands Received 1 July 2002, and in revised form 12 September 2002
Abstract PML bodies are nuclear organelles that are associated with various diseases and are suggested to be involved in multiple cellular activities including transcriptional regulation, apoptosis, and antiviral defence. Because many proteins with different functions aggregate in PML bodies, it has also been suggested that these bodies function as nuclear depots. Some proteins consistently found in PML bodies may form a stable scaffold that regulates the recruitment of other proteins. Thus, some proteins might be stably integrated into PML bodies while others continuously exchange with the nucleoplasm. To study the dynamic properties of PML bodies and resident proteins, we constructed fusion proteins of Sp100, PML, and CBP with autofluorescent proteins. Using timelapse imaging, we show that PML bodies exhibit little movement but that small foci that contain Sp100 but not PML are dynamic and fuse with PML bodies. Furthermore, we show by monitoring fluorescence recovery after photobleaching that Sp100, PML, and CBP are dynamic components of PML bodies. This suggests that these proteins do not play a strict structural role in these bodies but that they function at other sites in the nucleoplasm. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: PML; Sp100; CBP; Time-lapse imaging; FRAP
1. Introduction Our view of how the cell nucleus is spatially and functionally organized has changed dramatically since it has become possible to study the localization and kinetics of nuclear components in living cells. With the advent of probe and green fluorescent protein (GFP) technologies as well as live cell imaging methods, chromatin dynamics, RNA transport, and protein localization and kinetics have been extensively studied. These studies have revealed that chromatin can be highly dynamic (Gasser, 2002; Tumbar et al., 1999), that RNAs are transported throughout the interchromatin space (Dirks et al., 1999, 2001; Politz et al., 1999), and that proteins involved in diverse nuclear processes are highly mobile (Chen et al., 2002; Dundr and Misteli, 2001; Houtsmuller et al., 1999; Leung and Lamond, 2002; *
Corresponding author. Fax: +31-0-71-5276180. E-mail address: [email protected] (R.W. Dirks).
Misteli et al., 1997; Snaar et al., 2000). Many nuclear proteins reside in nuclear compartments and, by tagging these proteins with GFP, the dynamic behavior of these compartments could be analyzed by time-lapse microscopy. Cajal bodies, for example, were shown to move in the nucleoplasm and occasionally were shown to fuse to form larger bodies and to split from larger bodies (Platani et al., 2000; Snaar et al., 2000). The functions of most nuclear compartments have not been completely resolved yet and this is particularly true for PML bodies. PML bodies, also referred to as ND10 (nuclear domain 10) or PODs (PML oncogenic domains), range in size from 0.2 to 1 lm and are present in 10–30 copies per nucleus. They fluctuate in size and number during the cell cycle (Everett et al., 1999). Since the identification of Sp100 as a component of PML bodies (Szostecki et al., 1990), many other proteins have been found present in these compartments (Matera, 1999; Negorev and Maul, 2001). Because these proteins fulfill multiple biological functions, PML
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bodies have been implicated to play a role in various cellular processes including the control of chromatin or heterochromatin architecture (Seeler et al., 1998), regulation of apoptosis (Guo et al., 2000; Wang et al., 1998), transcription (LaMorte et al., 1998), MHC class I antigen presentation (Zheng et al., 1998), detection of foreign protein or protein–nucleic acid complexes and proteolytic degradation (Everett et al., 1997; Tsukamoto et al., 2000), antiviral defense (Ishov and Maul, 1996), and cell proliferation and tumor suppression (Salomoni and Pandolfi, 2002). It cannot be excluded, however, that the main or only function of PML bodies may be to regulate the recruitment and release of proteins in order to control their availability at nucleoplasmic sites other than PML bodies as suggested by Negorev and Maul (2001). In their view, PML bodies function as a nuclear depot (Maul, 1998). Essential for the assembly and stability of PML bodies are PML and its modification by the small ubiquitin-related protein SUMO-1 (M€ uller et al., 1998). Cells lacking PML or expressing PML– RARa and RARa–PML fusion proteins, as in acute promyelocytic leukemia cells, do not reveal PML bodies. Less well understood is the role of SUMO-1 modification in PML body assembly because studies using PML mutants that cannot be SUMO-modified provide contradictory results (Ishov et al., 1999; M€ uller et al., 1998). Therefore, SUMO modification has been proposed to be a control mechanism in the accumulation of proteins at PML bodies. For example, SUMO modification of PML proved essential for the recruitment of Daxx, a protein enriched in condensed chromatin, to PML bodies (Ishov et al., 1999). Also, other proteins, including CREB binding protein (CBP), Blooms (BLMs), and Sp100, do not localize at PML bodies in the absence of PML, though SUMO modification of PML does not seem to play a role in their recruitment. In contrast to PML, SUMO-1 modification of Sp100 does not appear to play a role in its deposition at PML bodies (Sternsdorf et al., 1997). How proteins are recruited to PML bodies is still enigmatic but adaptor proteins and protein–protein interactions are assumed to be essential to this process. On the basis of their kinetic behavior, PML and Sp100 are suggested to play structural roles in PML bodies (Boisvert et al., 2001). Fluorescence recovery after photobleaching (FRAP) analysis of GFP–PML and GFP–Sp100 movement revealed that these chimeric proteins are immobile inside PML bodies (Boisvert et al., 2001). At the same time, another PML inhabitant, the transcription coactivator CBP, was shown to move rapidly into and out of these bodies. The observation that PML and Sp100 are immobile in PML bodies is in contradiction to the hypothesis that nuclear compartments are the reflection of steady-state association/dissociation of its residents with the nucleoplasm (Phair and Misteli, 2000). In fact, proteins that localize at di-
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verse nuclear compartments were shown to exhibit rapid movements (Kruhlak et al., 2000; Phair and Misteli, 2000; Snaar et al., 2000). We therefore reevaluate the kinetic behavior of PML, Sp100, and CBP and show that all three proteins are dynamic components of PML bodies. In addition, using time-lapse fluorescence microscopy, we show that PML bodies exhibit little movement in interphase cell nuclei but that Sp100 foci that do not contain PML exhibit more extended movement. The generally small-sized Sp100-containing foci appear to fuse with larger PML bodies but not to separate from them. These observations suggest that homooligomerization of Sp100 in the nucleoplasm can occur in the absence of PML and precede recruitment to PML bodies.
2. Materials and methods 2.1. Cell culture VH10 human primary fibroblasts and U2OS human osteosarcoma cells were cultured on 3.5-cm glass bottom petri dishes (Mattek, Ashland, MA) in DMEM without phenol red containing 1.0 mg/ml glucose, 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 lg/ ml streptomycin and buffered with 25 mM Hepes buffer to pH 7.2 (all from Invitrogen). Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. 2.2. Plasmid constructs and cell transfection Full-length PML in an EGFP–C1 vector was a gift from Dr. A.G. Jochemsen (LUMC, Leiden). The EGFP fragment was replaced by ECFP and EYFP using the restriction sites AgeI and BsrGI. Sp100 cDNAs were amplified from total RNA isolated from U2OS cells using reverse transcription PCR and the forward primer 50 GCGCGCGGTACCATGGCAGGTGGGGGCCAG-30 containing the KpnI site and the reverse primer 50 GCGCGCCCCCGGCTAATCTTCTTTACCTGACCC-30 containing the XmaI site. The cDNA for CBP was PCRamplified from a plasmid containing full-length CBP (provided by Dr. E. Kalkhoven, LUMC, Leiden). Purified amplified PCR fragments were inserted in-frame into the KpnI–XmaI fragment of pEYFP–C1 and pECFP–C1 (Clontech Laboratories) and verified by sequencing. Transient transfections of cells were performed at approximately 60–80% confluence using DOTAP under conditions recommended by the manufacturer (Roche Diagnostics GmbH). 2.3. Immunofluorescence Cells grown on microscopic glass slides were washed in PBS and fixed with 4% formaldehyde in PBS for 5 min
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at room temperature. Subsequently, cells were permeabilized in PBS containing 1% Triton X-100 for 15 min and washed in Tris-buffered saline (TBS), pH 7.4. Cells were then incubated for 45 min at room temperature with the mouse anti-PML antibody 5E10 (Stuurman et al., 1992) diluted 1:10 in TBS containing 0.5% (w/v) blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany). Following three rinses in TBS containing 0.05% Tween, cells were incubated with the secondary antibody goat anti-mouse Alexa 594 (Molecular Probes, Eugene, OR, USA; dilution 1:1000) for 45 min, washed in TBS, and mounted in Vectashield (Vector Laboratories, Burlingame, California) containing 50 lg/ml 40 ,60 -diamidino-2-phenyl indole (DAPI). 2.4. Time-lapse microscopy Approximately 16 h after transfection, three-dimensional time-lapse images were recorded on a Leica TCS/ SP2 confocal microscope system. During imaging, the cells were kept in a closed chamber and the temperature of the culture medium was kept at 37 °C using a temperature-controlled ring (Harvard Apparatus), which was placed around the culture chamber, and a microscope objective heater (Bioptechs, Butler, PA). Image stacks of cells were acquired using a 100 NA 1.4PL APO lens and analyzed with Leica software. For each nucleus, 5–10 optical sections separated by 0.5 lm were acquired every 1–5 min over a period of 1–2 h. In total for each transfection experiment 10 nuclei were recorded. Images were viewed as maximum intensity projections of each time point. 2.5. Frap and flip FRAP and FLIP were performed on the Leica TCS/ SP2 confocal microscope system using spot bleaching. For FRAP experiments, 5–6 confocal sections were acquired followed by a single bleach pulse of 6 s in FRAP and multiple bleach pulses of 0.5–6 s/body or region in FLIP. Then, images of multiple sections were collected at time intervals of 20–60 s. Quantitative analysis of the amount of fluorescence in bleached regions relative to unbleached regions was performed with the software options delivered with the Leica system and with Excel. Corrections were made for background and for fluorescence fading that occurred during the measurements.
3. Results and discussion 3.1. Fusion proteins of PML, Sp100, and CBP with EYFP or ECFP localize to PML bodies PML, Sp100, and CBP were fused at their amino terminus to EYFP and to ECFP and transiently trans-
fected in VH10 fibroblasts and in U2OS cells. Before time-lapse experiments were started, the distribution of these chimeric proteins at PML bodies was confirmed by immunocytochemistry. EYFP– and ECFP–Sp100 and EYFP– and ECFP–PML were shown to localize at PML bodies in both cell lines. EYFP– and ECFP–CBP also localized at PML bodies, but generally at lower intensities, and the amount varied among cells. In addition, the CBP fusion proteins were shown to localize throughout the nucleoplasm, excluding nucleoli. These results show, in agreement with previous studies (Boisvert et al., 2001), that the GFP fusion proteins localize in the same manner as the endogenous counterparts. However, close examination of image stacks of cells containing EYFP–Sp100 revealed small Sp100 aggregates or foci that were not stained with the anti-PML antibody. The presence of foci that contain Sp100 but not PML became even clearer from cells that were double-transfected with EYFP–Sp100 and ECFP–PML or with ECFP–Sp100 and EYFP–PML. As shown in Fig. 1, these double-transfected cells allowed us to distinguish between PML bodies containing both PML (cyan in Fig. 1A) and Sp100 (yellow in Fig. 1B) and bodies that contain Sp100 only (small yellow bodies in Figs. 1B and C). Sp100 foci that do not contain PML have previously been interpreted as precipitates of Sp100 that could not be accommodated by PML bodies when Sp100 is extensively over expressed (Negorev et al., 2001). In this context, it has been suggested that the amount of binding sites for Sp100 in PML bodies would be limited and that Sp100 would not self-aggregate in these domains. These observations have been made, however, in fixed cells and do not exclude the possibility that multimerization of Sp100 may precede uptake by PML bodies. Interestingly, the N-terminal region of Sp100 (amino acids 33–149), which is important for dimerization, is also required for PML body targeting (Sternsdorf et al., 1999). Thus, it is very possible that homodimerization of Sp100 might be required to generate a functional PML body-targeting signal. Thus far, however, there is no experimental evidence that Sp100 might form multimers (Sternsdorf et al., 1999), though interactions with other proteins have been described. Therefore, it would be interesting to investigate whether other proteins than PML are present in Sp100 foci. Because HP1 is found present in PML bodies and is shown to interact with Sp100 (Seeler et al., 1998), it is a good candidate. 3.2. EYFP–Sp100 bodies are dynamic and fuse with PML bodies To test the idea that Sp100 may form aggregates in the nucleoplasm that have then the ability to fuse with PML bodies, we thought to perform time-lapse experiments using VH10 fibroblast as well as U2OS cells
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Fig. 1. Double expression of ECFP–PML and EYFP–Sp100 in U2OS cell. ECFP–PML (A) and EYFP–Sp100 colocalize in PML bodies (pink in C). A number of small Sp100-containing bodies can be distinguished that do not contain PML protein (yellow in C). Bar ¼ 10 lm.
transiently expressing EYFP–Sp100 to examine the dynamic behavior of Sp100-containing foci. For this purpose, cells containing low to moderate levels of EYFP–Sp100 have been visually selected on the basis of fluorescence intensity. The results revealed that the larger EYFP–Sp100 containing bodies, which were shown to correspond to PML bodies by immunocytochemistry, were relatively immobile while the smaller foci revealed significant movement. Notably, not all small-sized foci were dynamic during the time a cell was imaged. Mostly, two to five of the small-sized foci were shown to move during the time-span a cell was imaged (generally 2 h). Interestingly, the small EYFP–Sp100 foci were observed to fuse with larger bodies and occasionally with other small foci (Fig. 2). Muratani et al. (2002) recently reported the rapid movement of smaller PML bodies that were labelled with EYFP–Sp100. However, these bodies have been interpreted as PML bodies, while our
results show that these bodies do not necessarily contain PML and as such should not be named PML bodies. Our results suggest that Sp100 has the ability to form homomers in the nucleoplasm outside PML bodies and that the resulting aggregates or foci can fuse with PML bodies. At present, it is not known what drives the fusion of bodies or which interactions are involved to ‘‘store’’ Sp100 in PML bodies. To answer the last question, we are currently studying protein–protein interactions with possible candidates using fluorescence energy transfer (FRET) and fluorescence live cell imaging microscopy (FLIM). 3.3. Sp100, PML, and CBP are dynamic components of PML bodies Having shown that Sp100 foci can fuse with PML bodies, we addressed the question of whether Sp100
Fig. 2. Time-lapse images of VH10 cell expressing EYFP–Sp100. As indicated by arrows, small EYFP–Sp100 bodies move in the cell nucleus and fuse with larger PML bodies.
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would be a dynamic component of PML bodies. For this purpose, cells were transfected with EYFP–Sp100 and subjected to photobleaching. Because the level of expression of tagged Sp100 could affect the extent to which they move we selected cells expressing moderate levels of EYFP–Sp100. By using the spot bleach mode of the Leica confocal system, an optical section was scanned and 1–3 PML bodies containing EYFP–Sp100 were selectively marked. The intensity of the laser light was adjusted that in the selected areas, the fluorescence intensity was dropped by more than 90% following photobleaching during 6 s. Images were then collected each 20–60 s to follow the entry of unbleached EYFP– Sp100 into PML bodies. Recovery of fluorescence was already noticeable within 1 min after bleaching and nearly complete recovery of fluorescence was ob-
tained within 7 min (Fig. 3). This suggests that there is a continuous exchange of EYFP–Sp100 in PML bodies. We then repeated the FRAP procedure for cells expressing EYFP–PML or EYFP–CBP. EYFP–PML revealed kinetic behavior similar to that of EYFP–Sp100 (Fig. 4). A much more rapid exchange of EYFP–CBP was observed. Within 10 s after photobleaching, a full recovery of fluorescence of EYFP–CBP in PML bodies was observed, suggesting that CBP is highly dynamic in the cell nucleus. Also FLIP, experiments in which PML bodies or selected areas in the nucleoplasm outside PML bodies were bleached for extended periods revealed that CBP is highly dynamic. Already within 15 s, continuous photobleaching of a small marked area resulted in nearly complete loss of nuclear fluorescence (Fig. 5). Furthermore, fluorescence intensities within and outside
Fig. 3. FRAP of three PML bodies in a VH10 cell expressing EYFP–Sp100. The bleached areas are indicated by circles. Images are taken before (A), just after (B), and at regular time points after bleaching (C and D show examples). The fluorescence intensities in the bleached and in three unbleached areas were measured and expressed as relative intensities (after correction for background). The mean intensity values of the intensities measured in the bleached regions were calculated during the time-lapse experiment, corrected for fluorescence fading, and shown in a fluorescence recovery curve (E). This curve shows that full recovery of EYFP–Sp100 is obtained after 7–8 min.
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Fig. 4. FRAP of a PML body in a VH10 cell expressing EYFP–PML. The first image is taken before the marked PML body is bleached (A), the second just after it is bleached (B), and the third and fourth images are examples of time points collected after bleaching (C and D). The fluorescence intensity in the bleached region was measured each 20 s for 9 min, corrected for background fluorescence and for fluorescence fading, and expressed as relative intensities (E). The fluorescence recovery curve shows that full recovery of fluorescence is obtained after 8–9 min.
PML bodies were shown to decrease at the same rate. Also, FLIP experiments confirmed that PML and Sp100 are dynamic components of PML bodies although, as shown for EYFP–Sp100 (Fig. 6), it took significantly more time to bleach all PML and Sp100. These results are consistent with the view that nuclear proteins travel throughout the cell nucleus and transiently interact with other proteins, which may lead to their appearance in nuclear bodies (Phair and Misteli, 2000). Clearly, the time of interaction may vary among different proteins, as became apparent from the different residence times of CBP, Sp100, and PML in PML bodies. Our observation that CBP is highly dynamic is in agreement with results presented by Boisvert et al. (2001). However, our observations that Sp100 and PML are also dynamic components of PML bodies, although to a lesser extent than CBP, are in clear contradiction to
their observations. They observed very little recovery of GFP–PML and GFP–Sp100 even after 10 min, when half of the cell nucleus was photobleached. On basis of these recovery times, it was concluded that GFP–PML and GFP–Sp100 form relatively immobile complexes in the nucleoplasm as well as within PML bodies and play a structural role in PML bodies. This discrepancy in results cannot be easily explained but we followed a different approach to bleach PML bodies. Rather than bleaching half-nuclei, we selectively bleached PML bodies using spot bleaching. The advantage of this method is that movement of GFP fusion proteins that reside in the near vicinity of PML bodies is taken into account, and not only molecules that have to travel over large distances. Probably more important for the interpretation of our results is that we kept our cells at 37 °C during all
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Fig. 5. Determination of FLIP of EYFP–CBP that is expressed in a U2OS cell. The circle in the image taken before bleaching (A) indicates the area that is continuously photobleached while images were collected each second. The time series shows that after 15 s nearly all EYFP–CBP present in this cell is photobleached (B–H).
measurements, while Boisvert et al. performed their measurements at room temperature (22 °C), which slows down energy-dependent processes. Indeed, when we performed our FRAP measurements of EYFP–Sp100 and EYFP–PML at room temperature, we measured significantly longer recovery times. After photobleaching EYFP–Sp100 or EYFP–PML in a PML body, full
recovery of fluorescence was observed only after 20 min. This indicates that the incubation temperature of the cells has a strong impact on the mobility of Sp100 and PML and possibly of nuclear proteins in general. Furthermore, this temperature effect suggests that uptake of SP100 and PML by PML bodies requires energy or, alternatively, that these proteins are transported
Fig. 6. FLIP in a VH10 cell expressing EYFP–Sp100. Cells were imaged before bleaching (A) and at regular successive time points each preceded by a 6-s bleaching period of the indicated region in B. Nearly all EYFP–Sp100 fluorescence is lost within 10 min (B–H).
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through the nucleoplasm by an energy-dependent mechanism. Concerning the formation of PML bodies, we can only speculate how they are assembled. Apparently, PML is essential in this process, while other proteins may reside in these bodies because they interact directly or via other adaptor proteins with PML. As we have shown, Sp100 and PML are dynamic components of PML bodies. This makes sense because Sp100 as well as PML were reported to function at chromatin, which is not found present in PML bodies. For example, it has recently been shown that PML interacts with histone deacetylases (HDAC) and represses transcription by recruiting HDAC to target gene promoters (Wu et al., 2001). Like PML, Sp100 has been implicated as a factor that plays a role in transcriptional regulation. It has been shown to play a role in transcriptional repression by interacting with HP1 (Lehming et al., 1998; Seeler et al., 1998, 2001) and more recently to play a role in transcriptional activation by interaction with the transcription factor ETS-1 (Wasylyk et al., 2002). Interestingly, electron spectroscopic imaging analyses have revealed that some PML bodies are surrounded by blocks of chromatin (Boisvert et al., 2000). It is temptive to speculate that the transcriptional activity of the genes located in these domains is under the control of protein factors present in PML bodies, such as Sp100, PML, and CBP. This, however, does not preclude the possibility that Sp100 and PML also have specific functions within PML bodies. At least, for Sp100 and PML to be active in various biochemical pathways intact PML bodies are required (Salomoni and Pandolfi, 2002).
Acknowledgments We thank Alt Zantema, Eric Kalkhoven, and A.G. Jochemsen for PML and CBP plasmids. This work was supported by the Dutch Scientific Organization NWO program ‘‘4D Imaging of Living Cells and Tissues’’, Grant 901-34-144.
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