Transbilayer Coupling of Lipids in Cells Investigated by Imaging Fluorescence Correlation Spectroscopy

Abstract

Plasma membrane hosts numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation of the plasma membrane is emerging as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the co-existence liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions. However, this phase/domain separation is nanoscopic and transient in live cells. It is recently proposed that transbilayer coupling between the inner and outer leaflets of the plasma membrane is driven by their asymmetric lipid distribution and by dynamic cytoskeleton-lipid composites that contribute to the formation and transience of Lo/Ld phase separation in live cells. In this Perspective, we highlight new approaches to investigate how transbilayer coupling may influence phase separation. For quantitative evaluation of the impact of these interactions, we introduce an experimental strategy centered around Imaging Fluorescence Correlation Spectroscopy (ImFCS), which measures membrane diffusion with very high precision. To demonstrate this strategy we choose two well-established model systems for transbilayer interactions: crosslinking by multivalent antigen of immunoglobulin E bound to receptor FcεRI, and crosslinking by cholera toxin B of GM1 gangliosides. We discuss emerging methods to systematically perturb membrane lipid composition, particularly exchange of outer leaflet lipids with exogenous lipids using methyl alpha cyclodextrin. These selective perturbations may be quantitatively evaluated with ImFCS and other high-resolution biophysical tools to discover novel principles of lipid-mediated phase separation in live cells in the context of their pathophysiological relevance.

Lipid Driven Inter-leaflet Coupling of Plasma Membrane Order Regulates FcεRI Signaling in Mast Cells

“…The observation of global changes of diffusion of both order- and disorder-preferring lipid probes after LEX resembles those we observed previously after antibody crosslinking and immobilization of CTxB-GM1 complexes (74). However, the degree of probe diffusion changes in these two treatments (LEX and GM1 crosslinking) are different.…”

“…3D and 3H). In contrast, antibody crosslinking of GM1 with anti-CTxB antibody results in only ∼16% reduction of D of these probes (74). LEX appears to cause changes in the order of the entire outer leaflet.…”

“…Thus, this difference is likely due to relatively larger inter-leaflet coupling effects (as monitored by inner leaflet probe diffusion) by LEX than antibody crosslinking of GM1. Overall, our group so far has experimentally evaluated various modes of ‘outside-in’ inter-leaflet interactions (this study and (12, 74)) and found primarily two different outcomes as measured from precise diffusion measurements of these inner leaflet probes: either stabilization of phase-like separation (e.g., Ag-clustering of IgE-FcεRI receptors (12) or global change of membrane order (LEX in this study) and antibody crosslinking of CTxB-GM1 (74)). This body of work establishes the power of our bootstrapping-based analysis of diffusion coefficients of lipid probes measured by ImFCS (12) to delineate subtle changes of membrane organization in live cells and thereby reveal underlying biophysical principles.…”