SMrT assay for real-time visualization and analysis of clathrin assembly reactions

Abstract

Clathrin-mediated endocytosis manages the vesicular transport of the bulk of membrane proteins from the plasma membrane and the trans-Golgi network. During this process, discrete sets of adaptor proteins recognize specific classes of membrane proteins, which recruit and assemble clathrin lattices on the membrane. An important determinant to the success of this vesicular transport reaction is the intrinsic ability of adaptors to polymerize clathrin on a membrane surface. Adaptor-induced clathrin assembly has traditionally been analyzed using static electron microscopy-based approaches. Here, we describe a methodology to follow adaptor-induced clathrin assembly in real-time using fluorescence microscopy on a facile model membrane assay system of supported membrane tubes (SMrT). Results from such assays can be conveniently run through routine image analysis procedures to extract kinetic parameters of the clathrin assembly reaction.