Exploring Cell Surface–Nanopillar Interactions with 3D Super-Resolution Microscopy

[Image: see text] 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.