DOI: 10.5281/zenodo.7460047
title: Receptor exocytosis imaged with high temporal resolution for diverse receptor cargos

description: Cells perceive and interact with their environment in part through the expression, activation, and regulation of receptors on their plasma membrane. These receptors are dynamically trafficked from the plasma membrane in a process called endocytosis and delivered to the plasma membrane via exocytosis. Different receptors take diverse routes through the cell before being delivered via exocytosis. The data in this project focuses on 3 prototypical plasma membrane receptors - the B2 adrenergic receptor, the µ opioid receptor, and the transferrin receptor. Using a pH-sensitive green fluorescent protein variant, we visualized these receptors in cells as they recycled to the plasma membrane. We subsequently hand-labeled a subset of the data in order to build an automated image analysis method that could be used to detect receptor exocytosis across diverse imaging conditions. This repository contains our primary microscopy data from these studies as well as the labeling for use in supervised machine learning. These data support Evans et al 2021 and subsequent publications. <strong>Data Collection</strong><br> TIFF image stacks were collected using a Nikon Eclipse TiE Inverted Microscope using TIRF illumination with a solid state 488nm laser through a Nikon 60x/1.49NA TIRF objective and captured using an Andor iXon 897+ EMCCD camera. The camera was windowed to a 300x300 pixel view and images were collected with a 18.5ms exposures (~54Hz). Images were collected across two days, with two coverslips of each condition collected on day 1, and one coverslip collected on day 2. <strong>DNA Constructs</strong><br> The 3 cargos imaged in these data are the transferrin receptor (TfR), the B2-adrenergic receptor (B2AR, B2), and the µ opioid receptor (MOR). Constructs encoding these receptors, tagged extracellularly with the ph-sensitive GFP variant Superecliptic pHluorin (SpH, Sankaranarayanan et al. 2000, have been previously described in Yudowski et al. 2006 for B2AR, Yu et al. 2010 for MOR, and Yudowski et al. 2009 for TfR. <strong>Cell Culture</strong><br> HEK293 cells were cultured in DMEM High Glucose (Hyclone) supplemented with 10% Heat Inactivated FBS (Gibco). Cells expressing B2 and MOR were stably selected from transient transfection using G418. Cells expressing TfR were transfected 3 days before the experiments presented here using Effectene following manufacturers' instructions. Before imaging, cells were transferred to 25mm diameter #1.5 glass coverslips (Electron Microscopy Sciences). Two days after plating, experiments began. <strong>Imaging conditions</strong><br> Cells were imaged in L-15 minimal media supplemented with 1% FBS. For MOR and B2, cells were imaged for 1 minute at ~0.16Hz without perturbation. Then agonist was added (10µM DAMGO for MOR, 10µM isoproterenol for B2) to the media and cells were imaged for 5 minutes to ensure that receptors clustered and internalized. After internalization, cells were bleached with 100% laser power for 1 minute and then imaged at 54Hz to visualize exocytic events. exocytosis was captured for up to 20 minutes after initial treatment, one cell at a time. For TfR, a single frame was taken before bleaching to show receptor expression levels and then cells were bleached and imaged as described above. <strong>Data blinding</strong><br> After collection, files were renamed as described in <em>map.md</em>. All metadata files and internalization imaging were separated into the 2 "extras" folders. The exocytosis movies were 'scrambled' to hide cargo identity using the included <em>scrambler.py</em> file. <em>OPP_scramble.log</em> described the mapping of scrambled filenames to the original imaging. <strong>Human labeling</strong><br> A subset of the images (22, with roughly equal representation across cargos) were hand labeled for exocytic events. Images were viewed in FIJI Schindelin et al. 2012 nad played back at 0.5x. When exocytic events were identified by eye, the playback was paused and the appearance of an event was found through manual advancing of the frames of the movie. The event was labeled using the Cell Counter plugin. Each movie was watched twice to identify as many events as possible. Labeled events are saved a <em>&lt;movie-name&gt;-ZYW-1.xml</em> in this dataset. <strong>Data organization</strong><br> All exocytic event movies and any matching human labeling are included in this base directory. All internalization movies and all metadata for all movies are included in the Extras folder for the day that movie was recorded. Coverslip and cargo identity are listed in <em>map.md</em> and the ground truth for cargo identity is in <em>OPP_scramble.log</em>