Influenza M2 Transmembrane Domain Interacting with Lipid Membranes: An Atomic Force Microscopy and Fluorescence Microscopy Study

Abstract

By clustering at domain edge of host cell membranes, M2 protein of influenza A virus was proposed to participate in virus budding and scission. Despite large interest, the molecular mechanism that is responsible for M2 enhanced membrane curvature is obscure. Previous studies have focused on a water-soluble helix located at the cytoplasmic side of M2. A recent study by Hong and coauthors showed that by adopting a flexible conformation, transmembrane (TM) helix can actively promote curved membrane structures (Yao et al., 2015, Proc Natl Acad Sci USA 112, 10926-10931). Their observation prompts us to ask whether the TM domain of M2 (M2TM) can play a similar role in generating raft dependent membrane curvature. We use high-resolution liquid-compatible atomic force microscopy (AFM) and fluorescence microscopy to study M2TM interacting with planar and vesicular lipid membranes. Specifically, we directly visualize molecular-level structure of M2TM in fluid lipid membranes. We also find that cholesterol and sphingomyelin play an important role in governing M2TM oligomerization. We next investigate M2TM distribution in lipid membranes containing liquid-ordered (Lo) and liquid-disordered (Ld) phases. We find that M2TM exclusively partitions into Ld phase. In addition to topographic information, our AFM measurements also entail mechanical properties of lipid membranes. Surprisingly, we find that M2TM increases bending stiffness of Ld phase, while leaving Lo phase unaltered. In our fluorescence microscopy study of giant unilamellar vesicles (GUVs), we show that M2TM enhances miscibility transition temperature of phase coexisting GUVs. In addition, we observe that M2TM can induce an array of vesicle shapes that have not been seen before. Together, our experimental data highlight that the TM domain of M2 can act as a lipid raft sensor and a membrane curvature enhancer.