title: Automated selection of regions of interest for intensity-based FRET analysis of transferrin endocytic trafficking in normal vs. cancer cells.
abstract: The overexpression of certain membrane-bound receptors is a hallmark of cancer progression and it has been suggested to affect the organization, activation, recycling and down-regulation of receptor-ligand complexes in human cancer cells. Thus, comparing receptor trafficking pathways in normal vs. cancer cells requires the ability to image cells expressing dramatically different receptor expression levels. Here, we have presented a significant technical advance to the analysis and processing of images collected using intensity based Förster resonance energy transfer (FRET) confocal microscopy. An automated Image J macro was developed to select region of interests (ROI) based on intensity and statistical-based thresholds within cellular images with reduced FRET signal. Furthermore, SSMD (strictly standardized mean differences), a statistical signal-to-noise ratio (SNR) evaluation parameter, was used to validate the quality of FRET analysis, in particular of ROI database selection. The Image J ROI selection macro together with SSMD as an evaluation parameter of SNR levels, were used to investigate the endocytic recycling of Tfn-TFR complexes at nanometer range resolution in human normal vs. breast cancer cells expressing significantly different levels of endogenous TFR. Here, the FRET-based assay demonstrates that Tfn-TFR complexes in normal epithelial vs. breast cancer cells show a significantly different E% behavior during their endocytic recycling pathway. Since E% is a relative measure of distance, we propose that these changes in E% levels represent conformational changes in Tfn-TFR complexes during endocytic pathway. Thus, our results indicate that Tfn-TFR complexes undergo different conformational changes in normal vs. cancer cells, indicating that the organization of Tfn-TFR complexes at the nanometer range is significantly altered during the endocytic recycling pathway in cancer cells. In summary, improvements in the automated selection of FRET ROI datasets allowed us to detect significant changes in E% with potential biological significance in human normal vs. cancer cells.
authors.affiliation: Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA.
authors.forename: Ronak
authors.initials: R
authors.lastname: Talati
authors.affiliation: Department of Mathematics, The College of Saint Rose, 432 Western Avenue, Albany, NY 12203, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Vanderpoel
authors.affiliation: Department of Mathematics, The College of Saint Rose, 432 Western Avenue, Albany, NY 12203, USA.
authors.forename: Amina
authors.initials: A
authors.lastname: Eladdadi
authors.affiliation: Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA.
authors.forename: Kate
authors.initials: K
authors.lastname: Anderson
authors.affiliation: Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA.
authors.forename: Ken
authors.initials: K
authors.lastname: Abe
authors.affiliation: Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA. Electronic address: barrosm@mail.amc.edu.
authors.forename: Margarida
authors.initials: M
authors.lastname: Barroso
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004285:Dogs; D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D012984:Software; D061250:Transferrins / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D061250:Transferrins
keywords: Förster resonance energy transfer (FRET); Image processing; Region of interest (ROI); Strictly standardized mean differences (SSMD); Transferrin (Tfn); Transferrin receptor (TFR)