Super-Resolution Imaging and Quantitative Analysis of Membrane Protein/Lipid Raft Clustering Mediated by Cell-Surface Self-Assembly of Hybrid Nanoconjugates.

Abstract

Super-resolution imaging was used to quantify organizational changes in the plasma membrane after treatment with hybrid nanoconjugates. The nanoconjugates crosslinked CD20 on the surface of malignant B cells, thereby inducing apoptosis. Super-resolution images were analyzed by using pair-correlation analysis to determine cluster size and to count the average number of molecules in the clusters. The role of lipid rafts was investigated by pre-treating cells with a cholesterol chelator and actin destabilizer to prevent lipid raft formation. Lipid raft cluster size correlated with apoptosis induction after treatment with the nanoconjugates. Lipid raft clusters had radii of ∼ 200 nm in cells treated with the hybrid nanoconjugates. Super-resolution images provided precise molecule location coordinates that could be used to determine density of bound conjugates, cluster size, and number of molecules per cluster.