Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS.
Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS.
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DOI：
10.1038/srep11454
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发表时间：
2015-06-29
影响因子：
4.6
通讯作者：
Eggeling C
中科院分区：
文献类型：
--
作者：
Andrade DM;Clausen MP;Keller J;Mueller V;Wu C;Bear JE;Hell SW;Lagerholm BC;Eggeling C
过去几十年的重要发现改变了我们对质膜组织的看法。具体来说，皮质细胞骨架已成为膜蛋白横向扩散的关键调节剂。细胞骨架依赖性区室化脂质扩散已经被提出，但这个概念仍然存在争议，因为迄今为止这种现象仅通过与单一技术（单粒子追踪）相结合的易产生伪影的探针来观察到。在本文中，我们首次使用微创荧光染料标记的脂质类似物直接观察活细胞质膜中的区室化磷脂扩散。这些观察是使用光学 STED 纳米镜与荧光相关光谱 (STED-FCS) 相结合进行的，这种技术可以在亚毫秒时间尺度上研究膜动力学，空间分辨率低至 40 nm。具体来说，我们发现区室化磷脂扩散依赖于皮质肌动蛋白细胞骨架，并且这种受限扩散直接依赖于F-肌动蛋白分支成核剂Arp2/3。这些发现提供了确凿的证据，证明 Arp2/3 依赖性皮质肌动蛋白细胞骨架在质膜的动态组织中发挥着关键作用，可能调节基本的细胞过程。
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.