Differences in Lateral Membrane Organization in Fibroblasts Expressing Low and High Levels of the Influenza Viral Protein Hemagglutinin

Abstract

Lateral organization in cell membranes is crucial for biological processes such as endocytosis, signaling, protein transport, membrane trafficking and viral infection. Hemagglutinin (HA) is an influenza viral envelope transmembrane protein which has been shown to be associated with the liquid ordered (lo) phase. Fibroblasts that constitutively express HA, referred to as HAb2 cells, were used to characterize the lateral organization of HA. Since HAb2 cells have cell-to-cell variability in the membrane density of HA, cells with low and high expression levels of HA containing more consistent densities of HA were also used. Fluorescence correlation spectroscopy (FCS), confocal microscopy and fluorescence photoactivation localization microscopy (FPALM) were used to characterize membrane organization after labeling cells with fluorescent probes and/or transfecting with either EGFP-HA or Dendra2-HA. Preliminary FCS results show that the diffusion of the liquid-disordered (ld) phase probe Lissamine Rhodamine DOPE is similar in cells with high and low HA expression levels i.e. the amount of HA present does not influence the diffusion time. Confocal microscopy was used to study the effect of HA expression level on the extent of phase separation observed after blebbing was induced by DMSO treatment. FPALM was used to obtain details about membrane organization at the nanometer length scale.