Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories

TLDR

Biological membrane organization spans many length scales, yet limited far‑field microscopy resolution hampers distinguishing models, as illustrated by the heterogeneously distributed influenza HA protein associated with debated cholesterol‑rich lipid rafts. Fluorescence photoactivation localization microscopy was used to image tens of thousands of HA molecules at ~40 nm resolution in live and fixed fibroblasts, and the dynamics of HA within clusters were quantified in live cells to obtain an effective diffusion coefficient. HA molecules form irregular clusters ranging from ~40 nm to many micrometers, matching electron microscopy, and these observations were interpreted using several established biological membrane models.

Abstract

Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One canonical example of a heterogeneously distributed membrane protein is hemagglutinin (HA) from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using fluorescence photoactivation localization microscopy, we are able to image distributions of tens of thousands of HA molecules with subdiffraction resolution ( approximately 40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from approximately 40 nm up to many micrometers, consistent with results from electron microscopy. In live cells, the dynamics of HA molecules within clusters is observed and quantified to determine an effective diffusion coefficient. The results are interpreted in terms of several established models of biological membranes.