title: Mechanism of polarized lysosome exocytosis in epithelial cells.
abstract: Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigate the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain. In cells lacking either the ubiquitous AP-1A or the epithelial-specific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia and suggest that AP-1 is a crucial regulator of this process.
authors.affiliation: Department of Ophthalmology and Visual Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI 53705, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Toops
authors.forename: Fernando
authors.initials: F
authors.lastname: Diaz
authors.forename: Jose Maria
authors.initials: JM
authors.lastname: Carvajal-Gonzalez
authors.forename: Diego
authors.initials: D
authors.lastname: Gravotta
authors.forename: Francesca
authors.initials: F
authors.lastname: Mazzoni
authors.forename: Ryan
authors.initials: R
authors.lastname: Schreiner
authors.forename: Enrique
authors.initials: E
authors.lastname: Rodriguez-Boulan
authors.forename: Aparna
authors.initials: A
authors.lastname: Lakkaraju
mesh_terms: D000199:Actins / Q000378:metabolism; D033961:Adaptor Protein Complex 1 / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology; D008247:Lysosomes / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells
chemical_list: D000199:Actins; D033961:Adaptor Protein Complex 1; D002784:Cholesterol; D002118:Calcium