Linking the Dynamics of Clathrin-Mediated Endocytosis with Membrane Shape Changes in Living Cells with Nanometer Axial Resolution

Abstract

Clathrin-mediated endocytosis (CME) is essential for cellular homeostasis. Thus, a precise understanding of the biophysical properties of vesicle formation and internalization is necessary. Due to fast dynamics, heterogeneity, and the number of proteins involved in CME, our current knowledge is limited and biased. While fluorescence microscopy provides information about the spatio-temporal dynamics of proteins, it lacks evidence for membrane bending. In contrast, electron microscopy shows membrane curvature, but it lacks dynamic insight. Here, by using Simultaneous Two-wavelength Axial Ratiometry (STAR), we link clathrin dynamics with membrane shape changes. STAR is a TIRF based microscopy technique we developed that takes advantage of the variance in penetration depth generated by different laser wavelengths. By imaging proteins tagged with the EGFP-iRFP fusion (STAR probe), we can generate the ratio of fluorescent intensity and convert it into axial information. The initial transition of plasma membrane from flat to curved and its correlation with clathrin arrival, during endocytosis, remains unclear. We report three different bending dynamics in Cos-7 cells transfected with clathrin light chain STAR probe. Most often clathrin accumulates along with curvature generation, or curvature starts forming shortly before clathrin arrival. Rarely, clathrin first assembles as a flat lattice prior to vesicle formation. Surprisingly, a quarter of de novo clathrin assemblies does not induce curvature. We hypothesize that the flat lattices might be caused by the absence of activated receptors, curvature sensing/inducing proteins, or vesicle maturation markers. To test this and to further describe the difference in initial curvature generation, we are developing a three-color STAR approach to integrate endocytic components with membrane shape changes. In doing so, we hope to unify the physical process of vesicle formation in clathrin-mediated endocytosis with its dynamics.