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Phosphoinositides play important roles in the regulation of protein recruitment at specialized membrane domains, protein activity, and membrane dynamics. Phosphoinositide-protein interplay occurs via multiple mechanisms and proteins associate with membranes through different binding patterns. Determinations of membrane-binding mode and membrane penetration depth of proteins in lipid bilayer are thus important steps in characterizing the molecular mechanisms of membrane-protein interactions. Here, we show two standard in vitro assays using liposomes, diphenylhexatriene (DPH) anisotropy, and fluorescence quenching by brominated lipids to determine membrane penetration of proteins into lipid bilayer. These methods will provide useful tools to study membrane-protein association and uncover molecular details of protein-lipid interplay, which are important for understanding biological functions of membrane-associated proteins and membrane dynamics.

A large proportion of proteins are expected to interact with cellular membranes to carry out their physiological functions in processes such as membrane transport, morphogenesis, cytoskeletal organization, and signal transduction. The recruitment of proteins at the membrane-cytoplasm interface and their activities are precisely regulated by phosphoinositides, which are negatively charged phospholipids found on the cytoplasmic leaflet of cellular membranes and play critical roles in membrane homeostasis and cellular signaling. Thus, it is important to reveal which proteins interact with phosphoinositides and to elucidate the underlying mechanisms. Here, we present two standard in vitro methods, liposome co-sedimentation and co-flotation assays, to study lipid-protein interactions. Liposomes can mimic various biological membranes in these assays because their lipid compositions and concentrations can be varied. Thus, in addition to mechanisms of lipid-protein interactions, these methods provide information on the possible specificities of proteins toward certain lipids such as specific phosphoinositide species and can hence shed light on the roles of membrane interactions on the functions of membrane-associated proteins.

Total internal reflection fluorescence microscopy enables to analyze the localizations and dynamics of cellular events that occur at or near the plasma membrane. Total internal reflection fluorescence microscopy exclusively illuminates molecules in the close vicinity of the glass surface, thereby reducing background fluorescence and enabling observation of the plasma membrane in the glass-attached cells with a high signal-to-noise ratio. Here, we describe the application of total internal reflection fluorescence microscopy to analyze the dynamics of caveolae, which play essential physiological functions, including membrane tension buffering, endocytosis, and signaling at the plasma membrane.