Live Cell Strategy for Detection of Curvature Dependent Sorting of Membrane Associated Proteins

Abstract

The curvature of cellular membranes varies a lot from organelle to organelle and within small subsections of the plasma membrane. A growing amount of evidence suggests that membrane curvature serves a purpose of regulating oligomerization, activity, membrane protein conformation, and recruitment of proteins. We have developed a novel live cell strategy for quantification of protein densities with regard to membrane curvature. This fluorescence-based technique takes advantage of a neuronal-derived cell line forming a high amount of filopodia of various diameters (and therefore various membrane curvatures) serving as a model for highly curved membranes in the cell. Relating the protein density to membrane curvature in the filopodia allows us to asses protein sorting as a function of membrane curvature in living cells. The assay was validated by comparing the membrane localization of the non-curvature sensing, transmembrane protein Aquaporin0, and two I-BAR containing proteins, IRSp53 and MIM, which interact with negatively curved membranes. Quantitative analysis revealed significantly different negative curvature sensing behavior by the Aquaporin0, IRSp53, and MIM. Furthermore, probing three isoforms of Ras GTPases (H-Ras, N-Ras and K-Ras) with this new technique revealed membrane curvature dependent sorting of all these three membrane-anchored proteins in living cells.