Bacher CP et al. (2004), 4-D single particle tracking of synthetic and p...

XB-ART-2697

BMC Cell Biol 2004 Nov 23;5:45. doi: 10.1186/1471-2121-5-45.

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4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin - poor tracks.

Bacher CP , Reichenzeller M , Athale C , Herrmann H , Eils R .

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The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei. We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 mum - wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data. Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

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Species referenced: Xenopus laevis
Genes referenced: actl6a vim

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	Figure 1. A) Nucleus of a SW13 cell stably transfected with an expression plasmid encoding for GFP-NLS-vimentin. B) SW13 cell nucleus containing microinjected 100 nm-microspheres. C) SW13 cell transfected with GFP-vimentin showing cytoplasmic particle formation. A', B', C'): Corresponding Hoechst 33342 chromatin stain, A", B", C") superimposed images.
	Figure 2. Screen shot of the image processing platform TIKAL. (top) Image shows a sample two-dimensional section through a nucleus with binarized nuclear particles (red) counterstained with Hoechst 33342 stain (green). Pull down menu exemplifies different tools for quantitative analysis integrated into TIKAL. Numbers indicate different nuclear particles reconstructed by 3D isosurface reconstruction (bottom). Computed tracks of nuclear bodies over time are displayed as spheres on a string in the multi-dimensional scene viewer.
	Figure 3. 4-D visualization of vimentin particle trajectories a) in the nucleus and b) the cytoplasm. Each color represents an individual vimentin body. The respective centers of masses are indicated as spheres. Tracks are symbolized by interconnecting lines. Arrows indicate the different kinds of diffusion (see Figure 4a). Major types of movement are indicated: I) confined diffusion; II) obstructed diffusion; III) normal diffusion; IV) directed motion.
	Figure 4. Different classes of mean square displacement of tracked nuclear vimentin particles. a) Mean squared displacement (MSD) of four representative modes of particle mobility (x-axis: acquisition time in seconds; y-axis: MSD). Roman numbers: I) confined diffusion; II) obstructed diffusion; III) normal diffusion; IV) directed motion (The numeration refers to indicated trajectories in Figure 3a). b) The four different mobility classes are represented by particle trajectories. The trajectories correlate to the numbers indicated by arrows in Figure 3a.
	Figure 5. Classification of particles into four groups of diffusional motion according to cellular localization and their calculated anomalous diffusion coefficient. a) nuclear vimentin particles, (b) microinjected nuclear 100 nm microspheres, (c) cytoplasmic vimentin particles.
	Figure 6. 4-D – tracking of vimentin particles in the nucleus. (a, b) particles with restricted movement (confined diffusion) c) corralled particle and corresponding trajectories. (red = vimentin bodies; green = Hoechst 33342 staining).
	Figure 7. Chromatin intensity analysis showing the preprocessing steps for the Hoechst 33342 images: a) unprocessed original image; b) Gaussian smoothing; c) image classified into 8 regions of grey values; d) localization of particles in the cell nucleus (red); e) measuring the mean grey value intensity around the center of mass for each individual vimentin particle (see Methods for a complete description).
	Figure 8. Correlation plots between mean grey values (green line) of chromatin densities and particle velocity (blue line) over a time range of 20 minutes. Arrows indicate significant changes in intensity and particle velocity. Red: NLS-GFP-vimentin; green: Hoechst 33342 stain.
	Figure 9. Mobility of nuclear vimentin bodies under the influence of inhibitors. Classification was performed according to the diffusion coefficient. a) control; b) azide / deoxyglucose (10 mM / 50 mM); c) cytochalasin D (1 μg/ml); d) nocodazole (0.04 μg/ml); c) sorbitol (600 mM).

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Albrecht-Buehler, Reversible compression of cytoplasm. 1982, Pubmed