Dynamics of intra-cellular organization

We study intra-cellular trafficking in mammalian cells, focusing on the biosynthetic/secretory pathway and functions of the Golgi complex. For this purpose, we set up original tools to unravel the molecular mechanisms involved and to develop translational applications based on this knowledge.

Our work focuses on two aspects of cellular dynamics, the Golgi apparatus in the secretory and retrograde trafficking pathways, and the regulation of microtubule dynamics. Our studies are carried out using a variety of approaches such as live cell imaging to follow and quantify intracellular trafficking or microtubule dynamics in the presence or absence of disrupting agents (e.g. siRNA, mutants, small molecules). We are also involved in the development of novel approaches  and tools (e.g. High Content Screening, recombinant antibodies) in order to fully explore both the fundamental and applied aspects or our projects.

Figure 1 : Synchronization of protein secretion using the RUSH assay

The Golgi apparatus – We are interested in the mechanisms that provide structural and functional homeostasis to the Golgi apparatus. We studied its role in cell organization, its mode of inheritance during cell division and we identified novel Golgi localised proteins. We have developed a method to specifically inactivate Golgi complexes in intact cells used it to study Golgi apparatus maintenance in interphase. We are now studying the multiple Golgi-intersecting pathways. To this end, we have developed a systematic transport assay, the RUSH assay, that enables to study and quantify the transport a large diversity of cargo in living cells (Figure 1). It is also usable for automated High Content Screening. We are using the RUSH assay in collaboration with the BioPhenics screening platform of the Institut Curie to characterize the many Golgi-dependent cellular routes and to identify specific inhibitors in chemical libraries that may be used for therapeutic applications.

Microtubule dynamics – We are also interested in the processes regulating microtubule dynamics. We studied for instance the role of certain types of CLIP (cytoplasmic linker proteins) proteins in the dynamic instability of microtubules. We originally showed that CLIP-170 is localized in a dynamic way to microtubule polymerizing plus ends and then studied CLIP-170-related protein involved in membrane trafficking. More recently, we selected an antibody sensitive to tubulin conformational changes when bound to GTP. This antibody allowed us to propose a new model for microtubule dynamic instability. We are now studying the role of microtubule in the control of the secretory pathway using the RUSH assay.

Figure 2: Selection of antibodies in vitro by Antibody Phage Display

Recombinant antibodies (Figure 2)- In the past, we used a library of scFv fragments obtained form the MRC (obtained from G. Winter, UK) but we now developed our own synthetic library based on a humanized nanobody scaffold with a diversity of 3. 10⁹ clones. In addition, we have simplified and improved our antibody production methods and set-up new selection protocols. Organizing dedicated platforms, we now bring this expertise to collaborative projects aimed at addressing specific fundamental cell biology questions or at exploring novel cancer diagnosis and therapeutics options.