3D VISUALIZATION OF NUCLEAR DAMAGES IN HISTONE H2B-GFP TAGGED HE-LA CELLS: POST-IRRADIATION IMAGING

Abstract

     Here we suggest visualization of the spatial nuclear changes using unique He-La cell line stably expressing histone H2B-GFP fused protein as a chromatin signature. To do this we offered Carl Zeiss LSM900 confocal microscope-based acquisition of Z-stacks, coupled with the 3D image reconstruction and rendering using ZEN and UCSF Chimera. Our recently displayed 2D images, showed that γ-irradiation leads to spatially very complex nuclear changes, so that one can easily defined that 2D approach is largely unable to yield precisely analyzed results. Meaning that in our experiments γ-irradiation in dose of 30 Gy leaded to the drastic nuclear deformations, followed by fragmentation and massive emergence of multi-nucleated cells, we resorted to only reliable analytical way, namely to 3D visualization and modeling approach. Our interest was spotlighted on the γ-irradiation induced nuclear and nucleolar changes due to: (i) the cell nucleus is the largest, highly ordered but dynamic cellular compartment, housing around 1.5 m DNA strand in common genome length. Although giant sizes complete genome is spatially precisely arranged being compacted inside confine of average 15-20 µm in diameter. Such a big sizes expose nuclear chromatin to natural but potentially damaging factors, particularly to γ-irradiation. Therefore, it is crucial to follow dynamics and degree of morphological damages, under permanent monitoring of nuclear viability; (ii) the common length of rDNA chromatin clustered as tandems of r-genes is also large enough to represent easily accessible target after being exposed to γ-irradiation. Because, the key function of the nucleolus, as a ribosome factory, plays central role in whole cellular metabolism, monitoring of related structural damages are particularly challenging.  
     Using 3D imaging/modeling hardware combined with UCSF Chimera we managed to describe in details specific nuclear/nucleolar damages aroused as a consequence of γ-irradiation with dose of 30 Gy. In fact, present work represented first attempt to show modern digital 3D visualization facilities in order to motivate young Georgian bio-medical scientist to use available software and other microscopy-based digital techniques for the living and fixed cell/tissue 3D/4D imaging. Importantly, all these facilities are locally available at “Open Platform for Advanced Microscopy” established in New Vision University “Scientific and Education Center by Carl Zeiss”.