Endophilin N-Bar Domain is Sorted by Membrane Curvature in a Solution Concentration-Dependent Manner

Abstract

Membrane curvature provides an active means to control spatial organization and activity of cells and is regulated and explored by a plethora of peripheral membrane proteins, including dynamin, as well as proteins containing BAR domains and amphiphatic membrane-binding helices. The protein endophilin has been shown to be involved in curvature-sorting phenomena during clathrin-mediated endocytosis in neuronal synapses. The mechanisms underlying its curvature-sorting are currently unclear. In addition to scaffolding effects contributed by its BAR domain and N-terminal hydrophobic helix membrane insertion, oligomerization likely contributes to membrane curvature sensing and generation. In order to enhance the biophysical understanding of membrane curvature sorting, we are using the following approach. Highly bent cylindrical membrane tethers are pulled from pipette-aspirated giant vesicles. Fluorescence images revealed that rat A1 endophilin N-BAR domains (E-N-BAR) preferentially partitioned onto the tethers rather than the low curvature vesicles. We quantified the sorting of E-N-BAR via image analysis of confocal microscopy tether cross section images. We found that curvature-sorting of E-N-BAR characteristically depends on aqueous solution protein concentrations. Ratiometric assessments of curvature-partitioning at two different solution concentrations furthermore revealed nonlinear trends in curvature composition coupling. We also developed a new thermodynamic curvature/composition coupling model to analytically interpret our measurements. In addition, our findings from fluorescence photobleaching recovery (FPR) measurements showed that diffusion coefficients of E-N-BAR on tethers decrease with increasing membrane curvature, revealing curvature-dependent molecular crowding consistent with our curvature sorting theory.