Membrane proteins are abundant in eukaryotic cells, comprising an estimated 30% of all genes [1]. They are key to many cellular functions including signaling, cell adhesion, motility, metabolism, and flux of metabolites through cells [2,3] and are thus subjects of intense scientific investigations. For many years, however, progress in membrane protein research has been relatively slow, especially when compared to studies of soluble proteins [4,5]. This is now rapidly changing, and our mechanistic understanding of membrane protein structure-function relationships is steadily growing [6].

The function of membrane proteins is often controlled by their association state, in homo and heterocomplexes. One of the best-known examples is the family of receptor tyrosine kinases (RTKs), which are single-pass membrane proteins that are inactive when monomeric but become active upon lateral association into dimers or higher order oligomers. Within the oligomeric complexes, the kinase domains are in close proximity, where they cross-phosphorylate each other and initiate downstream signaling. As a result, RTK function can be tuned through changes in RTK lateral association [7]. For other membrane proteins, the functional effects due to protein-protein interactions can be more subtle but nevertheless highly significant. For instance, the seven transmembrane helix G protein–coupled receptors (GPCRs) can signal as monomers while undergoing conformational changes in response to their ligands. Lateral interactions between GPCRs can impact the ligand-induced conformational changes and alter GPCR signaling [8, 9, 10]. In addition, membrane receptors can organize themselves into spatially enriched domains, leading to heterogeneous spatial distributions in the membrane. Within the enriched domains, the receptors may engage in either direct receptor-receptor interactions or indirect interactions that are mediated by lipids or cytoplasmic proteins [11]. The structural features of the enriched domains (also referred to as clusters) are not well understood, but there is evidence of differential functioning of the receptors inside and outside the domains [12].

Recent work has provided many examples of membrane protein interactions in cellular membranes that have functional significance [13, 14, 15]. Studies which utilize fluorescence have been instrumental in identifying and quantifying these interactions as they allow us to interrogate the dynamics of membrane proteins in the context of the native cellular environment [16]. Here, we overview recent experimental studies of membrane protein interactions that rely on three broad classes of fluorescence methods: super resolution microscopy, fluorescence fluctuation techniques, and Förster Resonance Energy Transfer (FRET) (Figure 1).