The exocyst and regulatory GTP ases in urinary exosomes

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 ELV s), 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 ELV s. 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 ELV s) 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 ELV s. 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 GTP ases, are present in human urinary ELV s. Finally, in living MDCK cells, ELV s 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 ELV s.