Soutenance de François Lienard

The efficiency and robustness with which the cell reads the genetic information needed to perform a task suggests that its nucleus possesses a spatio-temporal organization propitious to accomplishing this feat. The scientific community has made great advances over the last thirty years in understanding this organization thanks to microscopic and genomic techniques. Nevertheless, even if the spatial organization of the nucleus is getting better understood, it remains difficult to study the links between the different scales and biological processes. The internal dynamics of the nucleus takes place on spatial scales ranging from nanometers to micrometers, and on temporal scales from tens of microseconds to several hundred minutes, all in a complex environment, justifying the fact that no single technique is perfectly suitable. The motivation for this project was therefore first to develop a complementary technique for studying the internal dynamics of the nucleus. We have chosen dynamic light scattering (DDL) as this method offers the possibility of following processes over wide time ranges on sub-micrometer scales, in a global and non-invasive way.

The principle consists in sending a laser beam into the nucleus of a cell and then detecting variations in the scattered light intensity. These variations originate from biological processes internal to the nucleus; they are the temporal trace of these processes and they are measured as correlation times. The difficulty in measuring these times arises from the non-stationary nature of the scattered intensity, due to the out-of-equilibrium aspect of the nucleus. To measure these correlation times, we have implemented a new analysis method called "Multi-scale Time-Laplace", based on the CONTIN algorithm.

Thanks to this method, we were able to analyze the internal dynamics of the nucleus of two cell lines: HeLa cells and F9 cells. We were particularly interested in the dynamics of the nucleus of a HeLa cell under different culture conditions. Our experiments show that the internal dynamics of the nucleus of a living cell can be classified into three dynamics: i) fast correlation times, 10-3-10-1 s, which are suspected to be associated with fundamental and active processes in the nucleus, ii) medium correlation times 0.1 - 1 s, potentially due to the diffusion of protein complexes or large objects, iii) times 10 - 200 s, probably due to the diffusion of chromatin fiber in the nucleus. We will see that these characteristics apply to both HeLa and F9 cell lines. Two findings emerge from our experiments: We observe variations in the intensity of correlation times during the cell cycle, but the evolution of these times seems to be dominated above all by cell culture conditions.