Mobile foci of Sp100 do not contain PML: PML bodies are immobile but PML and Sp100 proteins are not

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 time-lapse 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.

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, Dirks et al., 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; 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 μm 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 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 (Müller et al., 1998). Cells lacking PML or expressing PML–RARα and RARα–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; Müller 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 diverse 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.