Computational protocol: Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

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Protocol publication

[…] For microscopy, cells were grown in Synthetic Dextrose without tryptophan (to minimize autofluorescence) at 25°C until early log phase. Cells were attached to concanavalin A–coated coverslips, which were sealed to slides with vacuum grease (Dow Corning, Midland, MI). Imaging was done at room temperature using an Olympus IX71, Olympus IX81, or Nikon TE300 equipped with 100×/numerical aperture 1.4 objectives and Orca-100 or Orca II camera (Hamamatsu). Appropriate filter sets and neutral density filters were used during imaging, and images were acquired at rates of 0.5–1 frame/s. Simultaneous two-color imaging was done using an image splitter (Optical Insight) to separate the red and green emission signals to two sides of the camera sensor using 565-nm dichroic mirror and 530/30- and 630/50-nm emission filters. To excite GFP, we used a 488-nm argon-ion laser; for RFP, we used a mercury lamp filtered through a 575/20-nm filter. The excitation beams from these two light sources were combined using a beam splitter. Images of immobilized microbeads that fluoresce at both green and red wavelengths were captured and used to align the cell images. Z-stacks were acquired through the entire cell at 0.2-μm intervals. Images were collected using MetaMorph software (Molecular Devices, Sunnyvale, CA) and processed using ImageJ (National Institutes of Health, Bethesda, MD). Patch lifetimes were calculated from endocytic sites that assembled and disassembled during the movie. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications SPIM, Microscopic phenotype analysis
Organisms Saccharomyces cerevisiae
Chemicals Phosphatidylinositol 4,5-Diphosphate