Ph.D Thesis


Ph.D StudentNaor Tal
SubjectQuantifying Cell Cycle Dependent Chromatin Dynamics during
Interphase by Live 3D Tracking
DepartmentDepartment of Biomedical Engineering
Supervisor ASSOCIATE PROF. Yoav Shechtman


Abstract

The study of cell cycle progression and regulation is of key importance to our understanding of fundamental biophysics as well as aging and other disease mechanisms. While extensive knowledge exists regarding epigenetic mechanisms and the function of regulatory factors, some aspects of chromosome dynamics change during interphase are still elusive, due to the difficulties associated with live-cell experiments. Local chromatin movements are generally considered to be constrained and relatively consistent during all interphase stages, although recent advances in our understanding of genome organization challenge this claim. Here we use high spatio-temporal, 4D (x, y, z time) localization microscopy by point spread function (PSF) engineering and deep learning-based image analysis, for live imaging of mouse embryonic fibroblast (MEF 3T3) and MEF 3T3 with a double Lamin A Knockout (MEF LmnaKO) cell lines, to characterize telomere diffusion during the interphase. We detected varying constraint levels imposed on the chromatin, which are prominently decreased during G0/G1. Moreover, in the absence of Lamin A, these differences between the cell cycle phases were significantly reduced. The latter result may reflect dynamic nucleus compartmentalization maintained by Lamin A. The 3D chromatin motion in MEF 3T3 has also shown anisotropy; while the axial and lateral diffusion exhibited different cell-cycle-dependent motion pattern. Our 4D measurements of telomere diffusion offer an effective method to investigate chromatin dynamics and reveal cell-cycle-dependent motion constraints, which may be caused by various cellular processes.