Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains

Biological membrane functions are coupled to membrane curvature, the regulation of Which often involves membrane-associated proteins. The membrane-binding N-terminal amphipathic. helix-containing Bin/Amphiphysin/Rvs (N-BAR) domain of ampiliphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quantitative experimental characterization. We have measured, tube-pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR), bound to tubular membranes pulled from micropipet-aspirated giant vesicles. We observed that fluorescently labeled DA-N-BAR showed significantly higher protein density on tubes compared to the connected low-curvature vesicle membrane. Furthermore, we found the equilibrium tube-pulling force to be systematically dependent on the aqueous solution concentration of DA-N-BAR, thereby providing the first quantitative assessment of spontaneous curvature generation. At sufficiently high protein concentrations, pulled tubes required no external force to maintain mechanical equilibrium, in agreement with the qualitative spontaneous tubulation previously reported for amphiphysin.