title: Apical localization of inositol 1,4,5-trisphosphate receptors is independent of extended synaptotagmins in hepatocytes.
abstract: Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.
authors.affiliation: Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Maria Jimena
authors.initials: MJ
authors.lastname: Amaya
authors.affiliation: Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, Connecticut, United States of America.
authors.forename: André G
authors.initials: AG
authors.lastname: Oliveira
authors.affiliation: Department of Cell Biology, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Lena K
authors.initials: LK
authors.lastname: Schroeder
authors.affiliation: Department of Cell Biology, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Edward S
authors.initials: ES
authors.lastname: Allgeyer
authors.affiliation: Department of Cell Biology, Yale University, New Haven, Connecticut, United States of America; Department of Biomedical Engineering, Yale University, New Haven, Connecticut, United States of America; Kavli Institute for Neuroscience, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Joerg
authors.initials: J
authors.lastname: Bewersdorf
authors.affiliation: Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, Connecticut, United States of America.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Nathanson
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000032:analysis* / Q000378:metabolism; D008297:Male; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D050857:Synaptotagmins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D053496:Inositol 1,4,5-Trisphosphate Receptors; D050857:Synaptotagmins; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium