Structural study of the endophilin A isoforms and their role in clathrin-independent endocytosis

Details

Supervisors

Pierre Morsomme

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Among the wide variety of membrane bending proteins, the superfamily of BAR domain proteins has been one of the most extensively studied in the recent years. BAR stands for Bin/Amphiphysin/Rvs167 which are the first three described proteins of this superfamily. These proteins have been shown to function in many cellular processes requiring local membrane deformation. One of particular interest is endocytosis, the process by which cells internalize their nutrients but also recycle their plasma membrane components. Many BAR domain proteins were shown to act in different stages of endocytic processes. In particular, members of the endophilin A BAR subfamily held our attention for their strong involvement in these mechanisms. The endophilin A subfamily is composed of three isoforms, endophilin A1, A2 and A3. They all share a common structure composed of four distinct domains: a N-terminal amphipathic helix, a BAR domain, a SH3 domain and a linker region connecting the last two. This global structure makes endophilins A remarkable proteins to build endocytic vesicles. Indeed, endophilin A were shown to be involved in well-described endocytic mechanisms such as clathrin-mediated endocytosis or fast endophilin-mediated endocytosis. In addition, recent research in my lab highlighted a new endocytic modality in which the A3 isoform plays a central role, but neither the A1 or A2 isoforms are involved. The first identified cargo of this new mechanism is the activated leukocyte cell adhesion molecule (ALCAM), also called CD166. The involvement of endophilin A3 in this mechanism but not of A1 and A2 raised the question of the origin of this isoform specificity. To answer this question, we developed a strategy composed of four distinct steps. We started with the obtention of a library of plasmids encoding for mutant proteins fused with GFP and constructed using a domain swapping approach with endophilin A2 and A3. We then used these plasmids to assess the colocalization between CD166 and the mutants overexpressed in HeLa cells. We next asked whether the overexpression of these mutants could rescue CD166 endocytosis in HeLa cells previously knocked-down for endophilin A3. Finally, we performed pull-down experiment with GST fused with CD166 cytosolic tail as bait protein to highlight potential interaction between CD166 and the mutants. The results from the three experiments all confirmed the specificity of endophilin A3 for CD166 endocytosis unlike endophilin A2. The linker-SH3 region appeared crucial for cargo recognition and the linker region’s variability between endophilin isoforms appeared to be not sufficient to explain the specificity of endophilin A3 in CD166 endocytic mechanism. Unfortunately, further investigation are still required to conclude exactly which domain(s) of endophilin A3 provide(s) its specificity for this mechanism.