Functional Imaging Of Nuclear Architecture (FIONA)

Our group studies the physical mechanisms governing the dynamics organization of chromatin and proteins in vivo. The nucleus contains one of the most intriguing molecule, DNA, as well as millions of proteins. Far from being structures with a static organization inside living cells, these molecular assemblies are constantly formed and unraveled according to their functional needs. How our genome fits into a nucleus around 200 000 times smaller than unwrapped DNA? How millions of proteins diffuse, find their target and assemble into condensates to perform their biological function within the proper time and space window? Our research focuses on these exciting questions using a multi-scale approaches combining super-resolution microscopy, advanced image analysis, genetics, micro-fluidic.

Our research is organized into 3 themes:

Dynamics organization of chromatin upon DNA damage

Dynamics organization of the nucleus in cells under pressure

Phase separation as an nuclear organizer

Experimental approaches:

We use single molecule microscopy (PALM - Photo Activable Localization Microscopy, STORM – Stochastic Optical Reconstruction Microscopy and Single Particle Tracking – SPT) to visualize and quantify the behavior of individual molecules in vivo. We use budding yeast as a model system allowing powerful genetic manipulation, as well as human cells.

Dynamics organization of chromatin upon DNA damage

Chromatin mobility is strongly altered in response to DNA damage in budding yeast and in some mammalian cells. The figure summarizes the different effects of DSB on chromatin dynamics (Figure from Miné-Hattab & Chiolo 2020, art by Olga Markova): i) damaged loci explore a larger nuclear volume during HR in diploid budding yeast, likely facilitating homology search; i) undamaged chromatin also becomes more dynamic during DSB repair, albeit to a lesser extent than repair sites. Such global change shows that increased chromatin mobility is a general response following DSBs, and not only an intrinsic property of homologous pairing, iii) multiple repair sites cluster and iv) DSBs relocalize to specific sub-nuclear compartment when the lesion occus in DNA regions that are difficult to repair.

We study the mechanisms governing these changes in chromatin mobility upon DNA damage, and their consequences.

Dynamics organization of the nucleus in cells under pressure

Most of the studies addressing nuclear organization have been conducted in exponentially growing cell cultures, and cell imaging is then performed on cells grown or arranged as a monolayer. In these conditions, there is still some growing space available and there is no particular mechanical stress. However, in nature, cells often have to proliferate in a confined environment. External mechanical stress can dramatically alter essential functions of the cell. In this research axis, we investigate how nuclear organization is altered by compressive stress and what are the consequences for genome integrity.

Phase separation as an nuclear organizer

The cell nucleus contains membrane-less condensates inside which specific proteins are more highly concentrated than elsewhere in the nucleus. This enhanced concentration is hypothesized to help the proteins coordinate and collectively perform their function. The formation of such foci at the right place in the nucleus, and within the proper time window, is essential for the functioning of the cell. However, how such membrane-less sub-compartments are formed, maintained or disassembled remains unclear. Importantly, the (de)regulation of these sub-compartments is tightly linked with the formation of protein aggregates related to the outbreak of neurological diseases. Several models are intensively debated in the literature to understand the physical nature of sub-compartments(figure from Miné-Hattab & Taddei 2019, art by Olga Markova). Among them, an attractive hypothesis is that membrane-less compartments arise from liquid phase separation and form droplets. Although some biochemical and wide field microscopy data support this hypothesis, these observations are at the limit of the optical resolution. In this research axis, we use single molecule microscopy approaches to distinguish between several models of condensates.