Exploring Cell Surface-Nanopillar Interactions with 3D Super-Resolution Microscopy.
Exploring Cell Surface-Nanopillar Interactions with 3D Super-Resolution Microscopy.
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DOI：
10.1021/acsnano.1c05313
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发表时间：
2022-01-25
期刊：
影响因子：
17.1
通讯作者：
Moerner WE
中科院分区：
文献类型：
--
作者：
Roy AR;Zhang W;Jahed Z;Tsai CT;Cui B;Moerner WE
质膜形貌已被证明强烈影响许多细胞过程的行为，如网格蛋白介导的内吞作用，肌动蛋白重排等。最近的研究已经使用三维（3D）纳米结构，如纳米柱，以压印明确定义的膜曲率（“纳米生物界面”）。在这些研究中，蛋白质及其相互作用是通过二维荧光显微镜探测的。然而，这种方法的低分辨率和有限的轴向细节对于确定沿着100 nm直径物体的蛋白质的相对空间位置和分布不是最佳的，这低于光学衍射极限。在这里，我们介绍了一种通用的方法来探索蛋白质的纳米级分布在纳米生物界面与10-20 nm的精度使用3D单分子超分辨率（SR）定位显微镜。这是通过结合硅油浸没物镜和3D双螺旋点扩散函数显微镜实现的。我们仔细调整物镜，以尽量减少石英纳米柱和细胞之间的球面像差。为了验证3D SR方法，我们对表面标记的纳米柱的3D形状进行了成像，并将结果与电子显微镜测量结果进行了比较。转向细胞中的跨膜锚定标签，高质量的3D SR重建显示膜紧紧地包裹在纳米柱周围。有趣的是，参与网格蛋白介导的内吞作用的细胞质蛋白AP-2在1/R（半径的倒数）膜曲率的特定阈值以上沿纳米柱沿着积累。最后，我们观察到AP-2和肌动蛋白优先积累在正高斯曲率附近的柱帽。我们的研究结果建立了一个通用的方法来研究纳米级分布的蛋白质在纳米生物界面使用三维SR显微镜。
Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used three-dimensional (3D) nanostructures such as nanopillars to imprint well-defined membrane curvatures (the “nano–bio interface”). In these studies, proteins and their interactions were probed by two-dimensional fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano–bio interface with 10–20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone-oil immersion objective and 3D double-helix point spread function microscopy. We carefully adjust the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R (the reciprocal of the radius) membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano–bio interface using 3D SR microscopy.
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