Reconstitution of clathrin-coated pits imaged at the single molecule level
Clathrin-mediated endocytosis fulfills crucial physiological roles in the uptake of cell surface adsorbed cargo that includes hormones, transferrin, immunoglobulins, LDL and many viruses.
Clathrin-coated pits (and the final product, coated vesicles) can be considered as a multi-protein machinery that in a highly coordinated manner excises selected portions of the plasma membrane that includes the selective capture of cargo. While the core architecture of this machinery is well studied and accessory regulatory components have been identified, significantly less is known about the molecular mechanisms regulating the growth of the clathrin lattice responsible for the formation of the coated pit. This study focuses on understanding the role of the endocytic accessory proteins termed ‘rim proteins’, that include Eps15, FCHO1/2 and intersectin, during the transition from early to the middle and late stages in the growth of the coated pit, a final stage defined by a deeply invaginated membrane with high curvature.
To this end we observe formation of synthetic clathrin-coated vesicles at the single molecule level in a microfluidic setup on supported lipid bilayer and pre-formed liposomes. By high spatio-temporal TIRF imaging and single molecule intensity calibration, arrival of single clathrin triskelia at initiation sites and growth of individual clathrin-coated pits is monitored. To evaluate the effect of accessory proteins on kinetics, initiation rate and efficiency of clathrin assembly, a dedicated analysis software is developed. Furthermore, we use calibrated single molecule detection to determine the stoichiometry of protein complexes involved in clathrin-mediated endocytosis using single molecule pulldowns.