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

Débora M Andrade,Veronika Mueller,Christian Eggeling,Congying Wu,B Christoffer Lagerholm,Stefan W Hell,Jan Keller,James E Bear,Mathias P Clausen

doi:10.1038/srep11454

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.

Highlights

Contrary, different methods have shown that the lateral motion of membrane molecules is constrained by different mechanisms
SPT experiments have suggested that even phospholipid diffusion in the plasma membrane is constrained, presumably by the cortical actin cytoskeleton[2,18]
In view of these findings, the “picket-fence” model was proposed[3]. This model hypothesises that direct anchoring of transmembrane proteins to cortical cytoskeletal filaments directly beneath the plasma membrane create restrictive barriers, and that these barriers indirectly constrain the diffusion of other membrane proteins and of lipids (Fig. 1a)

Summary

Introduction

Contrary, different methods have shown that the lateral motion of membrane molecules is constrained by different mechanisms. Previous STED-FCS investigations in live PtK2 and HeLa cells have further shown that diffusion of this DPPE analogue is mainly free and hardly hindered by transient interactions with other membrane molecules such as slow-moving or cytoskeleton-anchored proteins or by membrane curvature[5,23,24]. For these reasons, we believe that this lipid analogue is a very good candidate for delineating the effect on phospholipid diffusion due to cortical cytoskeleton-dependent membrane partition from that due to other hindrances. The current studies show for the first time that STED-FCS enables the observation of compartmentalised lipid diffusion, and that even minimally invasive phospholipid probes are spatially constrained within compartments of the plasma membrane

Methods

Results

Conclusion