title: A targeted RNAi screen identifies factors affecting diverse stages of receptor-mediated transcytosis.
abstract: Endosome transport by transcytosis is the primary mechanism by which proteins and other large cargo traverse epithelial barriers in normal tissue. Transcytosis is also essential for establishing and maintaining membrane polarity in epithelia and other polarized cells. To identify novel components of this pathway, we conducted a high-throughput RNA interference screen for factors necessary for the bidirectional transcytosis of IgG by the Fcγ receptor FcRn. This screen identified 23 genes whose suppression resulted in a reproducible decrease in FcRn-mediated transcytosis. Pulse-chase kinetic transport assays on four of the top-ranking genes (EXOC2, EXOC7, PARD6B, and LEPROT) revealed distinct effects on the apical and basolateral recycling and transcytotic pathways, demonstrating that these pathways are genetically separable. We also found a strong dependence on PARD6B for apical, but not basolateral, recycling, implicating this cell polarity gene in assembly or maintenance of the apical endosomal system. This dataset yields insights into how vesicular transport is adapted to the specialized functions of differentiated cell types and opens new research avenues into epithelial trafficking.
authors.affiliation: Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115.|Graduate Program in Biophysics, Harvard University, Cambridge, MA 02138.
authors.forename: Bradlee
authors.identifier: 0000-0002-2381-2170
authors.initials: B
authors.lastname: Nelms
authors.affiliation: Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115.
authors.forename: Natasha Furtado
authors.identifier: 0000-0001-5646-1744
authors.initials: NF
authors.lastname: Dalomba
authors.affiliation: Division of Gastroenterology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115 wayne.lencer@childrens.harvard.edu.|Harvard Digestive Diseases Center, Boston, MA 02115.
authors.forename: Wayne
authors.identifier: 0000-0001-7346-2730
authors.initials: W
authors.lastname: Lencer
mesh_terms: D000818:Animals; D016764:Cell Polarity; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D061985:Madin Darby Canine Kidney Cells; D010641:Phenotype; D021381:Protein Transport; D034622:RNA Interference; D011961:Receptors, Fc / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D057900:Transcytosis; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C470182:EXOC2 protein, human; D015395:Histocompatibility Antigens Class I; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C100688:Fc receptor, neonatal