Nanoscale membrane organization: where biochemistry meets advanced microscopy

Abstract

Understanding the molecular mechanisms that shape an effective cellular response is a fundamental question in biology. Biochemical measurements have revealed critical information about the order of protein-protein interactions along signaling cascades but lack the resolution to determine kinetics and localization of interactions on the plasma membrane. Furthermore, the local membrane environment influences membrane receptor distributions and dynamics, which in turn influences signal transduction. To measure dynamic protein interactions and elucidate the consequences of membrane architecture interplay, direct measurements at high spatiotemporal resolution are needed. In this review, we discuss the biochemical principles regulating membrane nanodomain formation and protein function, ranging from the lipid nanoenvironment to the cortical cytoskeleton. We also discuss recent advances in fluorescence microscopy that are making it possible to quantify protein organization and biochemical events at the nanoscale in the living cell membrane.

Figure 1. Biochemical principles regulating partitioning and nanoscale organization of membrane proteins

The formation of membrane nanodomains originating from lipid-lipid, lipid-protein and protein-protein based interactions implies the existence of a variety of biochemical principles that allow these interactions to occur at the molecular level. Proteins associated with cellular membranes have molecular determinants that allow them to be embedded in the highly hydrophobic milieu of the lipid bilayer. Several forms of lipid-based modifications provide the proteins either permanently or transiently with the right membrane anchor. Non-lipid modifications further contribute to the fine-tuning of receptor function and subsequent signal transduction. The same protein can undergo different modifications, however the regulation and interplay of these modifications are still unknown.

Figure 2. Mapping the membrane with NSOM

(A) Representative dual color excitation/detection NSOM image of LFA-1 integrin nanoclusters (red) and GPI-APs (green) at the cell surface of fixed monocytes in the absence of ligand. (B) The cartoon shows how the distance between the center of mass of a fluorescent spot and its nearest neighboring spot is calculated. Nearest inter-domain distance distributions of LFA-1 nanoclusters to its closest GPI-AP (bars) together with simulations of random spatial distribution of LFA-1 nanoclusters with respect to GPI-APs (red). The inset corresponds to the difference (i in %) between experimental data and simulations. At shorter distances (cross-over point in i) both distributions are significantly different with P 0.01. These results demonstrated that LFA-1 nanoclusters, known to co-cap with large raft domains, are in fact spatially segregated but proximal to GPI-AP hotspots. Reproduced with permission from van Zanten et al (108).

Figure 3. Capturing EGFR dimerization events

Tracking of QD585-EGF-EGFR (green) and QD655-EGF-EGFR (magenta) complexes. Trajectory over time shows close proximity of the two ligand-bound receptors with correlated motion. Insets: Stills from the acquired time series show moments of high colocalization and times when the receptors separate. Top right: Plot of distance between the two receptors as a function of time demonstrates fluctuations in separation. This is captured by the 3-state HMM that identifies repeated transitions (orange line) between dimer (D) and domain confined (C) states. Image courtesy of Shalini Low-Nam and similar to Low-Nam et al (80).