Computational protocol: The clathrin heavy chain isoform CHC22 functions in a novel endosomal sorting step

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Protocol publication

[…] Cells grown on coverslips were washed in warm PBS, fixed in warm PFA (3.7% in PBS, 10 min), then washed (2x, 5 min, PBS), permeabilized (4 min, 3% BSA, and 0.5% Triton X-100 in PBS), and blocked in blocking solution (3% BSA in PBS, 30 min). Antibody labeling was performed by inversion of coverslips on 20 µl blocking solution with primary or secondary antibodies (1–5 µg/ml), DAPI, or TO-PRO3 on parafilm and washing with PBS. Samples were mounted in the Prolong Antifade kit (Invitrogen). Images presented in ; ; Fig. S1, F and G; and Fig. S2, B and E were acquired by confocal laser scanning microscopy using a microscope system (EZ-C1Si; Nikon) equipped with a Plan-Apo 100x, 1.40 NA oil objective and 405-, 488-, and 561-nm laser lines. All other samples were analyzed using a microscope system (TCS SP5; Leica) with a 63x HCX Plan-Apo CS oil objective using 488-, 543-, and 633-nm laser lines. Imaging was performed at room temperature using Nikon immersion oil and Leica Type F immersion oil. DAPI, Alexa 488, Alexa 555, Alexa 568, Alexa 647, and TO-PRO3 fluorescence was sequentially excited using lasers with wavelengths of 405 (DAPI), 488 (Alexa 488), 543 (Alexa 555), 561 (Alexa 568), and 633 nm (Alexa 647, TO-PRO3). Images (1024 × 1024 pixels) were saved as TIFF files in NIS Elements software (Nikon), MetaMorph software (MDS Analytical Technologies), and Leica LAS AF software, and input levels were adjusted in Adobe Photoshop. Labeling detected in individual channels is presented in black and white in individual panels. 3x enlarged merged images of boxed areas are presented in color with labeling colors being color coded as described in accompanying figure legends. Overlap of red and green staining is yellow, overlap of red and blue staining is magenta, overlap of green and blue staining is turquoise, and overlap of all three channels is white.Image quantification was performed using National Institutes of Health’s ImageJ (http://rsb.info.nih.gov/ij). Colocalization of markers on vesicles in the periphery of double-labeled cells was performed after excluding the dense concentration of vesicles in the Golgi region because measuring individual vesicles is problematic in this area. Exclusion was the same for all cells and defined by a circular area of 72.2 µm2 centered at the most dense point of the Golgi. For each cell, individual marker fluorescence was measured in separate channels, and signals were adjusted to their dynamic ranges. For each marker, only particles larger than 0.094 µm2 (16 pixels) were automatically selected and counted. These automatically selected vesicles from individual labelings were then overlaid and colocalization was highlighted using the Colocalization Highlighter plugin (Pierre Bourdoncle, Institut Jacques Monod, Service Imagerie, Paris, France). The colocalized signal was again subjected to automatic particle count using the same automatic size exclusion as used to select individually labeled vesicles. The quotient of colocalized particles over total particles for one marker was calculated for individual cells and averaged.Degree of overlap of markers in individual cells was determined by Pearsons’ correlation coefficients and pixel shifts () to estimate colocalization as well as its specificity. Measurements were performed on total fluorescence of single cells using the JACOP plugin ().To quantify peripheral and perinuclear fluorescence of CI-MPR and STxB, individual cells were analyzed using the radial profile plugin (Paul Baggethun; ; see also illustrative Fig. S4). Fluorescent intensities within individual cells were measured as a function of distance from the centerpoint in a circular area of 2,927.7 µm2 (white circle in Fig. S4). This area was selected to include all the signals detected in all cells analyzed. The centerpoint for each cell was manually set as the center of the Golgi defined by the marker GM130. Data series were imported into GraphPad Prism software and the sum of all points were calculated using the Area under Curve function representing total fluorescence. Data points enclosed in an area of 182.5 µm2 around the centerpoint (red circle in Fig. S4) were defined as perinuclear with peripheral signal representing the difference between total and perinuclear signal. The boundary of the perinuclear area was set to include most of the Golgi staining for all cells analyzed. All calculations and graphs were performed and generated in Microsoft Excel and GraphPad Prism software. P-values were calculated using unpaired two-tailed Student’s t test. Figures were prepared in Adobe Photoshop and Illustrator software. Some data for this study were acquired at the Nikon Imaging Center at the Mission Bay campus of UCSF. […]

Pipeline specifications

Software tools MetaMorph, ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Heavy Chain Disease
Chemicals Glucose