Imaging FCS Delineates Subtle Heterogeneity in Plasma Membranes of Resting Mast Cells

Abstract

A myriad of transient, nanoscopic lipid-and protein-based interactions confer a steady-state organization of plasma membrane in resting cells that is poised to orchestrate assembly of key signaling components upon reception of an extracellular stimulus. Although difficult to observe directly in live cells, these subtle interactions can be discerned by their impact on the diffusion of membrane constituents. Herein, we quantified the diffusion properties of a panel of structurally distinct lipid-anchored and transmembrane (TM) probes in RBL mast cells by multiplexed Imaging Fluorescence Correlation Spectroscopy. We developed a statistical analysis of data combined from many pixels over multiple cells to characterize differences as small as 10% in diffusion coefficients, which reflect differences in underlying interactions. We found that the distinctive diffusion properties of lipid-anchored probes can be explained by their dynamic partitioning into ordered proteo-lipid nanodomains, which encompass a major fraction of the membrane and whose physical properties are influenced by actin polymerization. Effects on diffusion by functional protein modules in both lipid-anchored and TM probes reflect additional complexity in steady-state membrane organization. The contrast we observe between different probes diffusing through the same membrane milieu represent the dynamic resting steady-state, which serves as a baseline for monitoring plasma membrane remodeling that occurs upon stimulation.

A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane

“…In contrast to EFR3B-GFP, the transmembrane protein TMEM150A-GFP showed a much slower recovery (t1/2 = 55±13 s), with a smaller mobile fraction (~60%) (Figs. 2B-C), consistent with studies indicating that the diffusion of transmembrane proteins is generally slower than lipidanchored proteins [34,35] . As a control experiment, we found that the FRAP recovery properties of TMEM150A-GFP were similar to its homolog TMEM150B-GFP, which is structurally very similar but not involved in formation of the PI4KIIIα complex [20] (Fig.…”

“…The instrumental set-up, data acquisition and processing are described elsewhere [35] . Briefly, we collected time-lapse images (80,000 images at 3.5 ms frame rate) of ventral plasma membrane of fluorescently labelled RBL-2H3 cells using a home-built total internal reflection microscopy (TIRFM) equipped with high NA (1.49) oil-immersion objective and EMCCD camera (Andor iXon 897).…”

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

“…Phosphatidylcholines (PC) are present in both leaflets, and cholesterol flips rapidly between leaflets 5 . The outer leaflet lipids generally have more saturated chains than the inner leaflet yielding higher chain order and effective viscosity in the former, as shown by measurements of membrane order and diffusion 4,6,7 . The inner leaflet phospholipids have a net negative charge while the outer leaflet phospholipids are zwitterionic.…”

“…Work in our and other laboratories has shown that diffusion of membrane components can be measured to monitor interactions within each leaflet as well as coupling between the leaflets. This has been particularly explored in the context of phase-dependent behaviors 4,7,44,53 . Microscopic phase separation in synthetic giant unilamellar vesicles and GPMVs is readily observed by diffraction-limited fluorescence imaging 28,54 .…”

Bringing light to ER contacts and a new phase in organelle communication

“…To illustrate, we draw on our own investigations of the transmembrane immunoreceptor (FceRI) in the PM of mast cells, which binds antigen-specific immunoglobulin E (IgE) (6). In its homeostatic steady state, the PM is a heterogeneous distribution of lipids (and accompanying proteins) into nanoscale domains that resemble the liquid-ordered (L o ) and liquid-disordered (L d ) phases defined in model membranes (7). The term "raft" is associated with the ordered, L o -like domains.…”