title: An image analysis method to quantify CFTR subcellular localization.
abstract: Aberrant protein subcellular localization caused by mutation is a prominent feature of many human diseases. In Cystic Fibrosis (CF), a recessive lethal disorder that results from dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the most common mutation is a deletion of phenylalanine-508 (pF508del). Such mutation produces a misfolded protein that fails to reach the cell surface. To date, over 1900 mutations have been identified in CFTR gene, but only a minority has been analyzed at the protein level. To establish if a particular CFTR variant alters its subcellular distribution, it is necessary to quantitatively determine protein localization in the appropriate cellular context. To date, most quantitative studies on CFTR localization have been based on immunoprecipitation and western blot. In this work, we developed and validated a confocal microscopy-image analysis method to quantitatively examine CFTR at the apical membrane of epithelial cells. Polarized MDCK cells transiently transfected with EGFP-CFTR constructs and stained for an apical marker were used. EGFP-CFTR fluorescence intensity in a region defined by the apical marker was normalized to EGFP-CFTR whole cell fluorescence intensity, rendering "apical CFTR ratio". We obtained an apical CFTR ratio of 0.67 ± 0.05 for wtCFTR and 0.11 ± 0.02 for pF508del. In addition, this image analysis method was able to discriminate intermediate phenotypes: partial rescue of the pF508del by incubation at 27 °C rendered an apical CFTR ratio value of 0.23 ± 0.01. We concluded the method has a good sensitivity and accurately detects milder phenotypes. Improving axial resolution through deconvolution further increased the sensitivity of the system as rendered an apical CFTR ratio of 0.76 ± 0.03 for wild type and 0.05 ± 0.02 for pF508del. The presented procedure is faster and simpler when compared with other available methods and it is therefore suitable as a screening method to identify mutations that completely or mildly affect CFTR processing. Moreover, it could be extended to other studies on the biology underlying protein subcellular localization in health and disease.
authors.affiliation: Institut Pasteur de Montevideo, Montevideo 11400, Uruguay.
authors.forename: Lucilla
authors.initials: L
authors.lastname: Pizzo
authors.affiliation: Institut Pasteur de Montevideo, Montevideo 11400, Uruguay; IMERL, Facultad de Ingeniería, Universidad de la República, Montevideo, Uruguay.
authors.forename: María Inés
authors.initials: MI
authors.lastname: Fariello
authors.affiliation: Institut Pasteur de Montevideo, Montevideo 11400, Uruguay.
authors.forename: Paola
authors.initials: P
authors.lastname: Lepanto
authors.affiliation: Institut Pasteur de Montevideo, Montevideo 11400, Uruguay.
authors.forename: Pablo S
authors.initials: PS
authors.lastname: Aguilar
authors.affiliation: Institut Pasteur de Montevideo, Montevideo 11400, Uruguay; Instituto de Investigaciones Biotecnológicas Dr. Rodolfo A. Ugalde (IIB-INTECH), Universidad Nacional de San Martín, Consejo Nacional de Investigaciones Científicas y Técnicas (UNSAM-CONICET), San Martín, Buenos Aires, Argentina. Electronic address: akierbel@iibintech.com.ar.
authors.forename: Arlinet
authors.initials: A
authors.lastname: Kierbel
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000648:ultrastructure; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D061985:Madin Darby Canine Kidney Cells; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D015415:Biomarkers; C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: Apical; CFTR; Deconvolution; Image analysis