Imaging Sub-Diffraction Membrane Curvature Dynamics during Clathrin Mediated Endocytosis

Abstract

Proteins can generate and recognize curved membranes that in turn, modulate protein clustering, diffusion and biochemical reactions. The biophysical mechanisms of this dynamic relationship influence numerous biologically important events including receptor signal transduction, endocytosis, exocytosis and viral assembly. New approaches are needed to measure the dynamics of membrane bending in conjunction with protein recruitment and assembly. Here, I will discuss our work using back focal plane (BFP) spinning total internal reflection fluorescence (360-TIRF) microscopy to create uniform evanescent fields for quantitative imaging of molecular recruitment and assembly. We have paired these methods with BFP positioning for polarized-TIRF microscopy of membrane-oriented lipophilic fluorophores to image the sub-resolution membrane curvature. Using these methods, we have imaged the membrane bending dynamics during assembly of clathrin and dynamin at single endocytic sites in living cells. Our data demonstrate that this method is capable of imaging sub-resolution endocytic events and within the context of their surrounding topography. Together, these methods are enabling new insights into the interplay between assembly, reaction dynamics and membrane topography in living cells.