Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as cellular adhesion, clathrin-mediated endocytosis (CME) and actin rearrangement. Recent studies have investigated the molecular response of these processes through controlled variations in membrane topography by using vertical nanopillar substrates to precisely alter the membrane curvature of mammalian cells. In particular, 2D diffraction-limited fluorescence images of the interface between the cell and its underlying nanopillars (the Nano-Bio interface) reveal that specific proteins critical for CME accumulate at regions of high membrane curvature. However, the low resolution (around 250 nm) and limited axial detail of 2D diffraction-limited fluorescence images cannot determine the relative spatial position and organization of curvature-sensing proteins along the entire Nano-Bio interface. Here, we introduce a general method to explore the nanoscale organization of curvature-sensing proteins at the Nano-Bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. We first carefully optimize a silicone-oil objective with a correction collar to minimize spherical aberrations introduced by the refractive index mismatch between quartz nanopillars and the cell. The silicone-oil objective in addition with 3D double-helix point-spread function engineering methods are both used to image surface-labeled nanopillars in 3D. The negligible difference between the measured nanopillar diameters of our 3D SR reconstructions and SEM measurements confirm the high image quality of our reconstructions. Next, we tested our method with transmembrane labels. The high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. This approach can be used to measure the density and position of curvature-sensing proteins critical for CME such as clathrin and AP-2,  under conditions with various degrees of curvature. Our results establish a general method to investigate the nanoscale organization of curvature-sensing proteins at the Nano-Bio interface using 3D SR microscopy.<br/>

Exploring cell surface-nanopillar interactions with 3D super-resolution imaging

ACS Fall 2020 Virtual Meeting

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