Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS

Abstract

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It

“…In comparison, identically labeled phosphatidylethanolamine appeared to diffuse freely in the membrane (Eggeling et al, 2009), which implied that lipid-cytoskeleton interactions were not responsible for the anomalous cholesterol-dependent sphingolipid diffusion. Noteworthy, this finding of unhindered phosphatidylethanolamine diffusion conflicts with a previous single molecule tracking study (Fujiwara et al, 2002), and subsequent STED-FCS and single molecule tracking studies reported by these authors and others (Andrade et al, 2015; Fujiwara et al, 2016; Komura et al, 2016). Although, the authors of the STED study never concluded that the cholesterol-dependent trapping of sphingomyelin, GM1 and GPI-anchored proteins was indicative of tiny lipid rafts, their results were often cited by others as support for the lipid raft hypothesis (Lingwood and Simons, 2010; Levental and Veatch, 2016).…”

“…Perhaps the most influential super-resolution imaging studies of membrane organization revealed complex lipid dynamics that were ultimately inconsistent with partitioning into liquid-ordered membrane domains produced by favorable cholesterol-and sphingolipid interactions (Hiramoto-Yamaki et al, 2014; Honigmann et al, 2014; Andrade et al, 2015; Sevcsik et al, 2015). Stimulated emission depletion (STED) fluorescence microscopy imaging demonstrated fluorophore-labeled sphingomyelin, GM1, and a GPI-anchored protein were temporarily trapped within 20-nm-diameter areas in the plasma membrane of living cells, and this trapping was cholesterol-dependent (Eggeling et al, 2009).…”

Advances in Imaging Plant Cell Dynamics

“…5, F-I). A positive feature of STED is that, being a confocal microscopy-based technology, it can be used for superresolution in combination with quantitative imaging methods such as fluorescence correlation spectroscopy (FCS) either at the cell surface (Andrade et al, 2015) or in deeper parts of the cell (Lanzanò et al, 2017). Unlike SIM and PALM/STORM, which require postacquisition image processing to deliver the superresolved result, STED provides superresolution during acquisition.…”

Membrane shaping by actin and myosin during regulated exocytosis

“…A recent study suggested that not only proteins but also phospholipids are confined by the cortical actin network (Andrade et al, 2015). They used STED-FCS to monitor lipid probes in the plasma membrane and found that cortical actin networks induce spatiotemporal confinement of phospholipids on the plasma membrane.…”