Current Methods for Detecting Cell Membrane Transient Interactions

Abstract

Short-lived cell membrane complexes play a key role in regulating cell signaling and communication. Many of these complexes are formed based on low-affinity and transient interactions among various lipids and proteins. New techniques have emerged to study these previously overlooked membrane transient interactions. Exciting functions of these transient interactions have been discovered in cellular events such as immune signaling, host-pathogen interactions, and diseases such as cancer. In this review, we have summarized current experimental methods that allow us to detect and analyze short-lived cell membrane protein-protein, lipid-protein, and lipid-lipid interactions. These methods can provide useful information about the strengths, kinetics, and/or spatial patterns of membrane transient interactions. However, each method also has its own limitations. We hope this review can be used as a guideline to help the audience to choose proper approaches for studying membrane transient interactions in different membrane trafficking and cell signaling events.

Keywords: cell signaling; lipids; membrane biology; membrane probes; proteins; transient interactions.

Figure 1

Transient interactions control membrane functions. (A) During T-cell signaling, the MHCp ligand binding induces dynamic protein–protein interactions between T-cell receptor (TCR) and CD4. (B) The strength of lipid–lipid interactions regulates membrane phase separation and domain formation. White circles represent that lipids prefer ordered domain. The darker the blue shading of circles, the poorer the ability of these disordered phase-preferred lipids to pack tightly. (C) The correlation between membrane protein location and their interactions with lipids. Membrane lipids can be thus categorized into three groups: bulk, annular, and non-annular. (D) Dynamic cholesterol interaction with TCR regulates its activation and prevents non-specific responses.

Figure 2

The use of NMR and mass spectrometry for studying membrane transient interactions. (A) Dynamic membrane protein structural changes and interactions can be analyzed from the observed chemical shifts in 2D NMR spectrum. (B) Schematic of a general HDX-MS workflow to study protein conformation changes induced by dynamic lipid–protein interactions.

Figure 3

Fluorescence correlation spectroscopy measures membrane transient interactions at the single-molecule level. (A) Schematic of fluorescence cross-correlation of correlated and uncorrelated protein diffusions in the membrane. Increase in correlation among targets results in higher cross-correlation. (B) STED-FCS provides better spatial resolution to distinguish target interactions than the conventional confocal FCS.

Figure 4

TIRF-based single-molecule tracking to investigate membrane transient interactions. (A) Protein homodimerization and lipid–lipid interactions are transient process that can be studied with TIRF. (B) Transient interactions between proapoptotic BH3 protein and membrane-bound antiapoptotic Bcl-2 proteins can be studied using FLIM-FRET. FRET among Venus and mCherry facilitates Venus relaxation and lowers its lifetime.

Figure 5

Artificial membrane probes for studying dynamic membrane interactions. (A) Schematic of membrane two-hybrid assays for characterizing membrane protein–protein interactions based on the formation of deubiquitinating enzyme (DUB). (B) Schematic of applying photoactivatable lipid probes for proteome-wide mapping of membrane lipid–protein interactions. (C) Schematic of the toehold-mediated DNA displacement reaction for monitoring membrane lipid–lipid interactions. Reprinted with permission from You et al. (2017). Copyright (2017) Springer Nature Limited.