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


RESULTS
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.


CONCLUSIONS
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.
Bacher
Christian P
CP
Division of Theoretical Bioinformatics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. c.bacher@dkfz-heidelberg.de <c.bacher@dkfz-heidelberg.de>
Reichenzeller
Michaela
M
Athale
Chaitanya
C
Herrmann
Harald
H
Eils
Roland
R
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D000306:Adrenal Cortex Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018268:Adrenocortical Carcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000818:Animals; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics*; D042002:Chromatin Assembly and Disassembly / Q000235:genetics; D019295:Computational Biology / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods; D045586:Intranuclear Inclusion Bodies / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008863:Microspheres; D019913:Nuclear Localization Signals / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D014746:Vimentin / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics
D002843:Chromatin; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D014746:Vimentin; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate