Clausen M P, Colin-York H, Schneider F, Eggeling C, Fritzsche M
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, OX3 9DS Oxford, UK.
Department of Physics, Chemistry, and Pharmacy, MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
J Phys D Appl Phys. 2017 Feb 15;50(6):064002. doi: 10.1088/1361-6463/aa52a1. Epub 2017 Jan 11.
Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50 ± 15 nm and 120 ± 40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.
质膜与下方的皮质肌动蛋白细胞骨架之间的纳米级间距深刻地调节着细胞形态、力学和功能。测量这一距离一直是细胞生物学中的一项关键挑战。目前用于剖析纳米级间距的方法要么局限于使用固定样本的复杂测量设计，要么依赖于衍射极限荧光成像，其空间分辨率不足以量化纳米级的距离。在这里，我们使用双色超分辨率受激发射损耗（STED）显微镜克服了这一挑战，并准确测量了活的Jurkat T细胞中皮质肌动蛋白细胞骨架的密度分布以及肌动蛋白皮质与细胞膜之间的距离。我们发现皮质肌动蛋白密度分布不对称，平均宽度为230（+105/-125）nm。皮质与细胞膜的最大密度峰之间测得的空间距离呈双峰分布，平均值分别为50 ± 15 nm和120 ± 40 nm。结合皮质的有限宽度，我们的结果表明，在某些区域皮质肌动蛋白与细胞膜的距离小于10 nm，而在其他区域最大为20 nm。
