The Role of CLASPs in clathrin assembly on membrane

Abstract

Clathrin-mediated endocytosis (CME) internalizes most of the membrane proteins (cargo) in eukaryotic cells. Initiation, maturation and fission are the sub-stages during CME. Early events (initiation and maturation) during CME involve clathrin-associated sorting proteins (CLASPs) recruitment, membrane bending and clathrin assembly on plasma membrane. There are more than 100 molecules involved in CME including cargo, adaptor proteins (CLASPs), accessory proteins and clathrin. These CLASPs have weak affinity for specific cargo, PIP2, accessory proteins, AP2 and clathrin. These complex interactions are responsible for functional redundancy of these CLASP molecules and show little effect on CME in cells. Also limitation of specio-temporal resolution makes it difficult to understand precise role of individual CLASPs during clathrin assembly reaction. To address this we have used reconstitution approach by using defined set of protein molecules. Current assays and reconstitution schemes show minimal machinery required to form clathrin-coated pit on model membrane system but lack dynamics of the process. My work is divided into two parts, in the first part we have developed new assay system to study role of membrane curvature sensitivity of one of the indispensable adaptor molecules “epsin” in clathrin assembly. Second part has comparative study of different monomeric adaptor proteins in clathrin polymerization. Epsin is one of the essential CLASP molecules for clathrin-mediated endocytosis. We have developed a new assay system with budded supported membrane tubes (SMrT). Epsin shows membrane curvature sensitivity and sorts on highly curved membrane tubes in presence of phosphatidylinositol 4, 5-bisphosphate (PIP2). Fluorescently labeled clathrin clusters epsin molecules and assembles as foci on membrane tubes. We have shown the importance of membrane curvature sensitivity of epsin during clathrin assembly reaction. This assay system allows us to study real time clathrin polymerization dynamics on highly curve membrane tubes. Time constant for clathrin assembly on epsin coated membrane tubes is ~75 seconds, similar to time constant of clathrin-coated pit formation in cells. Using this assay system, we have compared clathrin assembly potential of 7 different alternative CLASP molecules using similar density on membrane surface. Out of these 4 CLASP molecules show clathrin assembly on supported bilayer tubes. We further did comparative analysis of clathrin polymerization rates across these adaptor proteins. This study will help us to understand the role of different CLASP molecules and their sorting during clathrin assembly reaction.