Asymmetric Membranes and the Study of Lipid Movement across Single Lipid Bilayers

Abstract

Fluorescence correlation spectroscopy (FCS) is one of the prominent tools to elucidate these dynamics in living cells but can only report on the dynamics at one given spatial position at a time. Using scanning fluorescence correlation spectroscopy (sFCS), we obtain a multitude of FCS measurements at different spatial locations. Here, we present a statistical analysis pipeline for sFCS data which allows for the accurate determination of the diffusion dynamics and the differentiation of free (Brownian) from hindered (non-Brownian) diffusion modes. We show free diffusion for phospholipids in model membranes and cells but reveal hindered diffusion of sphingolipids and GPI-anchored proteins in cells. Notably, these measurements can be performed using standard fluorescent dyes or proteins on a conventional confocal laser scanning microscope. To further investigate the dynamics on single cell level, we combine sFCS with stimulated emission depletion (STED) microscopy and by alternating conventional and super-resolved excitation we introduce line interleaved excitation scanning STED-FCS (LIESS-FCS). With LIESS-FCS the diffusion modes can be directly determined at multiple spots within the cellular plasma membrane providing detailed insights into organisation and function. Overall we are presenting a novel toolkit to investigate nano-scale molecular diffusion dynamics for shedding a new light on membrane organisation and heterogeneity. Ordered lipid domains (OLD) are thought to serve as signaling platforms. Here we show that their assembly does not involve intermediates in terms of nonregistered domains in the individual leaflets. The addition of photoswitchable lipids (ceramide PhoDAG-1) to the membrane forming lipid mixture allowed solubilizing and re-assembling OLDs by illuminating freestanding planar lipid bilayers at 365 nm and 475 nm, respectively. Differently colored fluorescent labels adopted a matching distribution in the two monolayers indicating that OLDs from the two leaflets were always in register. Tracing domain diffusion revealed perfect agreement with the Saffman-Delbr€ uck equation. The registration of domains as small as 10 nm in diameter is in line with the predicted role of line tension as a driving force for alignment (1). Registration of the observed micrometer sized domains is mainly driven by membrane undulations (2). 1. Galimzyanov, et al. Phys. Rev. Lett. 2015, 115:088101. 2. Galimzyanov, et al. Biophys. J. 2017. 1617-Plat Asymmetric Membranes and the Study of Lipid Movement across SingleThe great variety of lipid molecules in the cell membrane suggests their complex andunique role in cell function. The cell has further established unique lipid composition indifferent membranes within the cell for directed functionality. In addition, in membranes likethe plasma membrane (PM), there is an asymmetric distribution of lipids between the outer orexoplasmic and the inner or cytoplasmic leaflets and the physiological fate of cells depends onthe strict maintenance of this asymmetry. However, the exact ...