Plasma membrane asymmetry of lipid organization: fluorescence lifetime microscopy and correlation spectroscopy analysis

Anjali Gupta,Thomas Korte,Andreas Herrmann,Thorsten Wohland

doi:10.1194/jlr.d119000364

Abstract

A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.

Highlights

A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets
In support of these results, ITIR-FCS measurements on the same cell lines showed a higher diffusion coefficient and less domain-confined diffusion of lipid probes localized in the inner leaflet of the membrane and a slower diffusion accompanied with higher confinement in the outer leaflet of the membrane, with characteristic differences between cell lines
Our results reveal that the outer leaflet exhibits a liquid ordered environment, while the inner leaflet of the membrane is more similar to the liquid disordered phase

Summary

Introduction

A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. For outer leaflet membrane dynamics, we performed measurements at room temperature immediately after labeling cells with fluorescent lipid analogs at 4°C. We present the domain- and leafletspecific dynamics and organization of fluorescent lipid analogs in CHO-K1 and RBL-2H3 cell membranes, which are known to manifest distinct membrane properties and composition [40, 41].

Results

Conclusion