STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics

Abstract

Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality.

Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

“…It has been shown that biological membranes are heterogeneous at the nanoscale4344, and the size of lipid rafts could be smaller than tens of nanometers56. Considering the nano-sized subdomains of the L o phase observed in our experiments, molecular organization of rafts in cell membranes could be highly heterogeneous.…”

Lipid nanodomains change ion channel function

“…Results obtained from STED fluorescence correlation spectroscopy of live mammalian cells demonstrate local trapping of lipids in nanodomains for periods of up to 10 seconds. 37 Of particular interest for neurophysiology, we note that the proportion of cholesterol in nerve cell membranes is quite high, about 43% 38 and that cholesterol depletion in live mammalian cells inhibits both raft nanodomain formation and activation of the (PI(3)K/Akt) pathway. 8 The present findings finally demonstrate that nanodomains in cholesterol rich membranes also affect ion channel function.…”

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

“…Besides the spatial localization, the diffusion and interaction dynamics are also parameters essential to the function of Chol. FCS is a useful tool for investigating diffusion and interaction dynamics of fl uorescently labeled molecules (71)(72)(73)(74). In FCS, temporal fl uctuations in the detected fl uorescence due to molecular transits through the microscope's observation spot are analyzed to, for example, determine average transit times and molecular mobility.…”