This objective is being achieved through a variety of different imaging techniques to characterize the behavior of proteins within these subcompartments in the living cells nucleus. For example, various classes of nuclear bodies have been visualized by the expression of fluorescent protein-labeled component proteins, allowing their positioning and movement to be observed over time in living cells. This approach was used to demonstrate the energy-dependent movement of both PML and Cajal bodies within the nuclei of living cells and showed that Cajal bodies could merge and bud from one another (10,30). By imaging proteins labeled with different color fluorescent proteins, Platani and colleagues (32) showed that two different protein components, coilin and fibrillarin, were co-localized in the Cajal bodies. Importantly, this co-localization approach also revealed that the protein composition of the Cajal bodies changed over time (32). Thus, measurements of co-localization by multicolor imaging ((Figure 1)A) can supply important information about the molecular composition of these subnuclear domains.Figure 1.
These observations indicated that these nuclear bodies are active structures, and this was confirmed by measurements of the exchange of proteins within these subnuclear compartments using the technique of fluorescence recovery after photobleaching (FRAP). The FRAP technique uses photobleaching of the labeled proteins in a region of interest (ROI) to measure the kinetics of the redistribution of the population of fluorescent-labeled proteins over space and time ((Figure 1)B). The influx of labeled proteins from outside the bleached area is monitored, and plotting the recovery of fluorescence in the ROI provides an estimate of the mobile fraction of the labeled protein population ((Figure 1)B). It is important to note that proteins interact with varying affinity with other molecules in the cell, so diffusion constants determined from FRAP experiments must be carefully interpreted (33). The FRAP technique was recently used to monitor the flux of fluorescent protein-labeled component proteins through Cajal bodies. Studies by Sleeman et al. (34) showed that p80 coilin was rapidly exchanged from Cajal bodies. Dundr et al. (35) then analyzed many different proteins known to associate with Cajal bodies using a modified FRAP approach, and these studies revealed several distinct kinetic classes of protein exchange from these nuclear bodies. Together, these results demonstrated that these nuclear bodies are very dynamic structures.
A third quantitative imaging approach takes the analysis of subcellular structures in single cells to the cell population level. This approach uses computer algorithms to automate the detection and measurement of subcellular features in large sets of high-resolution images ((Figure 1)C). These automated approaches are designed to automatically segment images into ROI, and then apply the same set of rules to acquire measurements of those ROI in each image within the data set (20). These automated approaches are important because they allow consistent and rigorous analysis of subcellular features in each of the high-resolution images in the data set. We discuss in more detail the application of computer-based approaches to the analysis of nuclear protein distribution in the next section.The Selection of Transfected Cells for Image Analysis
Cell biologists are faced with a dilemma when using live-cell imaging to define the mechanisms that regulate the subcellular distribution of proteins. The problem is that the inherent heterogeneity in subcellular distribution prevents one from using the protein that is under investigation as the criterion for the selection of the cells to be imaged and analyzed. For example, the transcriptional corepressor proteins, including nuclear receptor corepressor protein (NCoR) and the silencing mediator of retinoid and thyroid hormone receptors, are organized with their histone deacetylase partners in discrete nuclear bodies called matrix-associated deacetylase bodies (36,37). Images of different cells transiently transfected with plasmids encoding NCoR labeled with yellow fluorescent protein (YFP) illustrates the heterogeneity in the organization of these subnuclear bodies, ranging from a diffuse distribution to an arrangement of highly concentrated focal bodies ((Figure 2)). This may partly reflect observations made by mRNA expression profiles that revealed extreme variability in transcriptional activity between individual cells in clonal populations; results that argue against the widely held notion of the “average” cell (38). Additionally, the heterogeneity in subnuclear organization may reflect cells that are in different phases of the cell cycle (25) and can be compounded further by differences in protein expression levels within the transfected cell population (39).