Computational protocol: Flat clathrin lattices: stable features of the plasma membrane

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[…] For single-channel dSTORM imaging, cells were grown in four-well-chambered coverglass slides (Thermo Fisher Scientific, Waltham, MA) to low confluency. Samples were rinsed in ice-cold phosphate-buffered saline (PBS) and fixed in 4% formaldehyde plus 0.02% glutaraldehyde on ice for 10 min, followed by a further 20-min incubation at room temperature. Autofluorescence was quenched with three 5-min incubations in PBS plus 50 mM glycine. Cells were blocked/permeabilized in 0.5% bovine serum albumin (BSA) plus 0.1% Triton X-100 for 30 min and then stained sequentially with primary and secondary antibodies for 1 h with gentle rocking, interspersed by three 5-min washes in blocking buffer. dSTORM imaging was performed on an IX71 inverted microscope (Olympus), fitted with a 100×/1.49 numerical aperture (NA) TIRF objective lens (Olympus), a 150-mW, 642-nm laser (TOPTICA Photonics, Victor, NY), and an iXon 897 electron-multiplying CCD (EMCCD) camera (Andor Technology, Belfast, UK). All components were controlled with custom software packages written in LabVIEW (National Instruments Corporation, Newbury, UK). Photoswitching buffer consisted of 1 μg/ml catalase, 50 μg/ml glucose oxidase, 40 mg/ml glucose, 12.5% glycerin, 1.25 mM KCl, 100 mM β-mercaptoethanol, and 1 mM TRIS-HCl (). Of importance, the imaging conditions were optimized to achieve the Nyquist sampling limit and ensure that continuous clathrin structures could be resolved.For dual-color PALM/dSTORM imaging, CHO-CCR5 cells were grown on 25-mm coverslips and transiently transfected with PAGFP-LCa 24 h before experiments. Cell surface CCR5 was prelabeled with MC5 conjugated to Alexa Fluor 647 for 1 h on ice in binding medium (RPMI 1640 without bicarbonate containing 0.2% BSA and 10 mM HEPES, pH 7.0), after which the samples were rinsed and allowed to equilibrate to 37°C for 20 min before treatment. After stimulation with 125 nM CCL5, samples were fixed and quenched as described before mounting in an Attofluor chamber (Life Technologies) for imaging on an ELYRA PS1 (Carl Zeiss AG, Oberkochen, Germany) fitted with a 100×/1.46 NA TIRF objective; 50-mW, 405-nm, 200-mW, 488-nm, and 150-mW 642-nm lasers; and an iXon 897 EMCCD camera.Single-channel dSTORM data were analyzed in rainSTORM (), and dual-color PALM/STORM acquisitions were analyzed in Zen 2011 (Carl Zeiss). Mean localization precision was 27.8 nm (±2.9) for dSTORM and 38.7 nm (±18.9) for PALM. Tetraspec beads (Life Technologies) were used as fiducial markers to monitor sample drift, correct chromatic offset (), and perform image registration. [...] We performed morphometric analysis of CCSs imaged by EM and superresolution microscopy. For EM analysis, large areas of membrane sheets were acquired by image tiling and stitching, individual clathrin structures were then identified visually and manually segmented by tracing their outlines in ImageJ (Research Services Branch, National Institutes of Health, Bethesda, MD) using a stylus and pad (Wacom Co., Kazo, Japan). Segmented structures were then used to generate binary images representing the distribution of clathrin across each membrane. For analysis by superresolution microscopy, dSTORM data were processed in rainSTORM and reconstructed as an image with a 25-nm pixel size. The CCSs in each acquisition were automatically segmented by applying a median filter with 3 × 3 pixel support and converting to a binary image. The binary data generated from EM or dSTORM images were then analyzed using the MATLAB Image Processing Toolbox (MathWorks, Natick, MA) to extract the size and shape characteristics of individual CCSs. Circularity was calculated from area and perimeter measurements using the standard circularity/roundness formula (4π area/perimeter2). […]

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