In this study we developed comprehensive bioinformatics tools to analyze the kinetic behaviour of small particles in the cell nucleus. For this purpose, fast time-lapse confocal laser scanning microscopy was used to record fluorescent particles in their chromatin environment. Automated image processing algorithms such as image registration and single particle tracking were instrumental to analyze the resulting complex data sets in a most efficient way. Usually, sophisticated image processing methods are widely not accessible for cell biology laboratories working with multi-dimensional data sets. A qualitative, interactive analysis of complex processes in living cells can yield interesting results. However, a quantitative insight into the underlying mechanisms can only be achieved by a rigorous computational analysis. While computational systems have been provided for estimating diffusion and binding constants based on photobleaching experiments of populations of small proteins [23
], integrated software packages for single particle tracking of nuclear bodies on the background of moving and shape changing objects have not been provided yet to the community. Here, our system TIKAL closes an important gap. For an automated analysis of even larger sets of spatio-temporal data as in this study any software system needs to be adapted to data storage systems that are devoted for handling such huge data sets [15
]. At the same time image analysis workflows have to be deployed onto computing clusters or the GRID ([34
], accepted). Both developments are underway in our laboratory.
We visualized the different kinetic behaviours of nucleus-injected 100 nm polystyrene microspheres. Furthermore, a stably transfected cell line expressing GFP-NLS-vimentin, which forms nuclear particles in the same size range as microspheres, was used. The majority of nuclear particles moved with obstructed diffusion within distinct corralled regions. This kind of movement was essentially found also for microinjected polystyrene beads. The obstructed diffusion behaviour supports the notion that these particles can diffuse within corrals restricted by dense chromatin regions. Upon chromatin remodelling distinct less dense chromatin regions are formed and enable the particle to move to an adjacent corralled region. We were able to quantitatively assess this phenomenon by measuring the chromatin intensity around an individual particle. Our data show that chromatin intensity decreases prior to a global velocity increase of the particle. Therefore we conclude that the particles do not actively push their way through the chromatin. Moreover, the chromatin itself is able to support or induce the movement of individual particles. The ability of the particle to move from one corral to the next is restricted and regulated by the surrounding chromatin remodelling activities. However, whether local chromatin regions can actively influence the destination of small nuclear particle movement has to be resolved in future investigations. With the present assay we cannot discern whether changes in the velocity of a body simply correlate with the entry of a body into a domain or whether the changes are caused by interaction between a body and the surrounding chromatin domain surfaces.
The addition of cellular inhibitors caused significant changes in the diffusional behaviour of nuclear particles. In all treatments a reduction of active transport processes were observed. This suggests that the coordination of nuclear processes such as chromatin remodelling is not solely dependent on single factors such as ATP, i.e. ATP consuming enzymes. Moreover, chromatin regions in interphase nuclei apparently move in a diffusional way [19
], while other factors such as cytoplasmic microtubules and actin filaments attached to the nuclear periphery possibly account for large-scale spatial chromatin rearrangements [35
The phenomenon of energy dependent nuclear body movement has been also described in other studies where an anomalous diffusion behaviour and an ATP- and transcription-dependent association of Cajal bodies with chromatin was reported [2
]. Further, upon ATP depletion in BHK cells rapid and large-scale movement of PML bodies stopped, whereas small localized movements of PML bodies were still observed [3
]. A recent examination of the dynamic behaviour of PML nuclear bodies showed their fission to microstructures after different physiological stresses, and their fusion upon recovery [36
]. Moreover it has been shown that movements of PML and other nuclear bodies can be described by diffusion of the individual body within a chromatin corral and its translocation resulting from chromatin diffusion [46
]. However, future systematic studies will help to reveal the influence of drug treatments and cellular inhibitors on the dynamic behaviour of those and other nuclear bodies.
A further interesting observation was the reversible formation of chromatin dense regions upon energy depletion. The general effect of this reorganization seems to influence the mobility distribution pattern of the particles only slightly. However, we could observe a significant decrease in directed motion of up to 30 % upon inhibition of energy-depended processes.
Furthermore, in order to evaluate the degree of nuclear particle movement with respect to cytoplasmic dynamics, we used the vimentin system to analyze cytoplasmic particle mobility. Mostly active transport processes were observed. Two possible explanations for this phenomenon are suitable. Vimentin, which belongs to the group of intermediate filaments, forms crossbridges to other cellular structures. Since the SW13 cells lack endogenous expression of intermediate filament proteins such as cytokeratins and vimentin, possible interactions with these cytoplasmic intermediate filaments can be omitted. Specific interactions with dynein have been described [37
]. Hence, newly synthesized vimentin is subjected to active transport processes and "guided" to cellular locations for the establishment of vimentin networks. Another explanation could assume that the filaments do not bind any cytoplasmic structure. In this less likely scenario the active transport of vimentin particles would result from the densely packed cytoplasm and the resulting pushing and pulling of adjacent actively transported molecules.
From our data we conclude that the NLS-vimentin system is very suitable for further studies of nuclear architecture. Though we obtained the same results with microinjection assays, the GFP-NLS-vimentin system has significant advantages such as the higher expression efficiency and the fact that the experiments can be performed in a cell system with normal proliferation characteristics.