title: The exocyst and regulatory GTPases in urinary exosomes.
abstract: Cilia, organelles that function as cellular antennae, are central to the pathogenesis of "ciliopathies", including various forms of polycystic kidney disease (PKD). To date, however, the molecular mechanisms controlling ciliogenesis and ciliary function remain incompletely understood. A recently proposed model of cell-cell communication, called "urocrine signaling", hypothesizes that a subset of membrane bound vesicles that are secreted into the urinary stream (termed exosome-like vesicles, or ELVs), carry cilia-specific proteins as cargo, interact with primary cilia, and affect downstream cellular functions. This study was undertaken to determine the role of the exocyst, a highly conserved eight-protein trafficking complex, in the secretion and/or retrieval of ELVs. We used Madin-Darby canine kidney (MDCK) cells expressing either Sec10-myc (a central component of the exocyst complex) or Smoothened-YFP (a ciliary protein found in ELVs) in experiments utilizing electron gold microscopy and live fluorescent microscopy, respectively. Additionally, human urinary exosomes were isolated via ultracentrifugation and subjected to mass-spectrometry-based proteomics analysis to determine the composition of ELVs. We found, as determined by EM, that the exocyst localizes to primary cilia, and is present in vesicles attached to the cilium. Furthermore, the entire exocyst complex, as well as most of its known regulatory GTPases, are present in human urinary ELVs. Finally, in living MDCK cells, ELVs appear to interact with primary cilia using spinning disc confocal microscopy. These data suggest that the exocyst complex, in addition to its role in ciliogenesis, is centrally involved in the secretion and/or retrieval of urinary ELVs.
authors.affiliation: Renal, Electrolyte and Hypertension Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Maria F
authors.initials: MF
authors.lastname: Chacon-Heszele
authors.affiliation: Renal Division, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina.
authors.forename: Soo Young
authors.initials: SY
authors.lastname: Choi
authors.affiliation: Renal Division, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Zuo
authors.affiliation: Renal Division, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina.
authors.forename: Jeong-In
authors.initials: JI
authors.lastname: Baek
authors.affiliation: Division of Nephrology and Hypertension, Department of Internal Medicine, University of Kansas Medical Center, Kansas City, Kansas.
authors.forename: Chris
authors.initials: C
authors.lastname: Ward
authors.affiliation: Renal Division, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina Department of Medicine, Ralph H. Johnson VAMC, Charleston, South Carolina.
authors.forename: Joshua H
authors.initials: JH
authors.lastname: Lipschutz
keywords: Cilia; ELV; ciliopathies; exocyst; polycystic kidney disease; urinary exosomes