Figure 1: Steps of endocytosis. Cargo sorting (1), lipid-membrane bending and bud formation (2), scission of transport-vesicles (3), release of transport-vesicles (4) and finally fusion of vesicles with the acceptor organelle.
Endocytosis is an essential process requiring orchestrated plasma membrane deformation for the uptake of nutrients from the surrounding cellular environment and turnover of plasma membrane components (Figure 1). Clathrin is the first coat protein involved in plasma membrane deformation identified over 50 years ago. However, the more recent discovery of clathrin-independent endocytic mechanisms led to the most challenging questions in the field: How are cargoes sorted in the absence of a clathrin coat and clathrin adaptor complexes? What are the proteins or complexes that link membrane curvature to cargo sorting?
Figure 2: Example of BAR domain protein. Adapted from McMahon & Gallop. 2005. Nature. 438 (7068), 590-596.
A part of the answer to these questions could be found in a fascinating superfamily of proteins (> 60 candidates in human genome) that are able to sense or induce membrane bending, and that could substitute clathrin and clathrin adaptors to complete endocytosis: BAR (Bin/Amphiphysin/Rvs) domain-containing proteins. Dimers of BAR domains are crescent-shape structures approximately 20 nm long and contain positively charged residues allowing interactions with the negatively charged head groups of phospholipids (Figure 2). They assemble to form highly organized oligomeric structures that are able to scaffold around tubular membranes. In addition to the BAR domain, these proteins often possess additional domains that allow binding to dynamin or to actin cytoskeleton regulators (SH3 domains), interaction with specific subsets of phosphoinositides (PX or PH domains), or the regulation of small GTPase or other signaling pathways (RhoGAP, RhoGEF, tyrosine kinase domains, etc.). This makes the superfamily of BAR domain proteins a pool of ideal candidates to connect membrane shape to actin cytoskeleton changes, small GTPase activity and regulation of other signaling pathways, or gene expression modulation. Some BAR domain proteins have already been involved in clathrin-dependent and independent processes, but there still remain a lot of questions.
My working hypothesis is that these cytosolic membrane curvature sensors/inducers BAR (Bin/Amphiphysin/Rvs) domain-containing proteins:
1°) play a functional role in endocytosis by connecting sensing/generation of membrane curvature to cargo sorting, particularly in clathrin-independent routes where there is no clathrin nor clathrin adaptors to fulfill these functions;
2°) play a pivotal mechanotransductional role as ‘signaling hubs’ connecting plasma membrane geometry and cargo selection to actin cytoskeleton remodeling, signaling pathways regulation, and gene expression modulation.
To unravel these functions of BAR domain proteins, my project aims to combine:
- advanced cell biology tools (Figure 3);
- various cutting-edge microscopy techniques (available in our institute/university or via collaborations such as with the group of Dr Ludger Johannes, Institut Curie, Paris, France);
- proteomics (available in our institute/university or via collaborations with the group of Prof. Ruddy Wattiez, UMons, Belgium);
- innovative biophysical approaches using model membranes (collaborations with the group of Dr Patricia Bassereau, Institut Curie, Paris, France), nanostructured cell culture surfaces (collaboration with the group of Prof. Christine Dupont, Institute of Condensed Matter & Nanosciences, Université catholique de Louvain), and atomic force microscopy combined with fluorescence imaging on live cells (collaboration with the group of Dr David Alsteens, Institute of Life Sciences, Université catholique de Louvain);
- cancer immunology (collaboration with the group of Prof. Pierre van der Bruggen, de Duve Institute, Université catholique de Louvain).
This should significantly help us to understand the functions of BAR domain proteins in endocytosis, and particularly 1°) how BAR proteins are recruited to curved plasma membrane in a physiological context, 2°) how they participate in cargo selection and specificity, and 3°) how they mediate downstream cellular effects in response to the membrane curvature cues. In addition, via interdisciplinary collaborations such as with immunologists, we aim to decipher the physiological significance of the endocytic mechanisms we discover or characterize in our lab. Our date might indeed be highly relevant in physiopathological contexts, as dysfunctions in some BAR domain proteins and endocytic mechanisms have been involved in genetic and neurodegenerative disorders, or cancers.
Figure 3: Example of protein (named X) identified in our lab as a new clathrin-independent cargo. In order to study the endocytosis of cargoes, we develop antibody uptake assays in our lab using various approaches such as flow cytometry. A-B, Kinetics of endocytosis followed by antibody uptake assay in flow cytometry. Cell surface disappearance of (A) cargo X and (B) biotinylated Transferrin (Tf) is measured on HeLa cells over time by flow cytometry and used to calculate the endocytosis rate (% of total). Remaining cell surface cargo X and Tf-biotin is detected with a fluorescently labeled specific antibody and streptavidin, respectively. Blue curves, siCtrl (Negative control siRNA). Red curves, siAP2M1 (µ2-Adaptin siRNA). C-F, Colocalization with µ2-Adaptin-mCherry over time. Anti-X (C) and Tf-Alexa488 (E) are bound to the surface of HeLa cells on ice and imaged at different time points of endocytosis after shift to 37°C. Colocalization percentage with µ2-Adaptin-mCherry is calculated from Manders’ coefficients and plotted over time for cargo X (D) and Tf-Alexa488 (F).
Past and present collaborators:
Here, I would like to acknowledge all the people I have or had the opportunity to collaborate/work with along the years.
- Institut Curie (Paris, France): Dr Ludger Johannes, Dr Patricia Bassereau, Dr Cécile Sykes, Dr Christian Wunder, Dr Christophe Lamaze, Dr Dhiraj Bhatia.
- Université catholique de Louvain (Belgium): Prof. Pierre Morsomme, Dr David Alsteens, Prof. Donatienne Tyteca, Prof. Patrick Gilon, Prof. Christine Dupont, Prof. Bernard Knoops, Prof. Pierre van der Bruggen.
- The Rockefeller University (NYC, USA): Dr Mijo Simunovic.
- Yale University (New Heaven, USA): Dr Joël Lemière.
- University College London (UK): Prof. Emmanuel Boucrot.
- Harvard University (Boston, USA): Dr Maria Daniela Garcia-Castillo.
- The University of New South Wales (Sydney, Australia): Dr Senthil Arumugam.
- Vanderbilt University (Nashville, USA): Prof. Anne K. Kenworthy.
- Institut Pasteur (Paris, France): Dr Anne A. Schmidt.
- Medical Research Council, Cambridge Biomedical Campus (UK): Dr Harvey T. McMahon.