title: Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy.
abstract: The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TAL cells is determined by exocytic delivery, recycling, and endocytosis. Surface biotinylation allows measurement of NKCC2 endocytosis, but it has low time resolution and does not allow imaging of the dynamic process of endocytosis. We hypothesized that total internal reflection fluorescence (TIRF) microscopy imaging of labeled NKCC2 would allow monitoring of NKCC2 endocytosis in polarized Madin-Darby canine kidney (MDCK) and TAL cells. Thus we generated a NKCC2 construct containing a biotin acceptor domain (BAD) sequence between the transmembrane domains 5 and 6. Once expressed in polarized MDCK or TAL cells, surface NKCC2 was specifically biotinylated by exogenous biotin ligase (BirA). We also demonstrate that expression of a secretory form of BirA in TAL cells induces metabolic biotinylation of NKCC2. Labeling biotinylated surface NKCC2 with fluorescent streptavidin showed that most apical NKCC2 was located within small discrete domains or clusters referred to as "puncta" on the TIRF field. NKCC2 puncta were observed to disappear from the TIRF field, indicating an endocytic event which led to a decrease in the number of surface puncta at a rate of 1.18 ± 0.16%/min in MDCK cells, and a rate 1.09 ± 0.08%/min in TAL cells (n = 5). Treating cells with a cholesterol-chelating agent (methyl-β-cyclodextrin) completely blocked NKCC2 endocytosis. We conclude that TIRF microscopy of labeled NKCC2 allows the dynamic imaging of individual endocytic events at the apical membrane of TAL cells.
authors.affiliation: Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan; Department of Physiology, Wayne State University, Detroit, Michigan; and.
authors.forename: Ankita Bachhawat
authors.initials: AB
authors.lastname: Jaykumar
authors.affiliation: Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan; Department of Physiology, Wayne State University, Detroit, Michigan; and.
authors.forename: Paulo S
authors.initials: PS
authors.lastname: Caceres
authors.affiliation: Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan;
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Sablaban
authors.affiliation: Experimental Therapeutics and Molecular Imaging Laboratory, Neuroscience Center, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
authors.forename: Bakhos A
authors.initials: BA
authors.lastname: Tannous
authors.affiliation: Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan; Department of Physiology, Wayne State University, Detroit, Michigan; and portiz1@hfhs.org.
authors.forename: Pablo A
authors.initials: PA
authors.lastname: Ortiz
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004705:Endocytosis / Q000502:physiology*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D064466:Solute Carrier Family 12, Member 1 / Q000378:metabolism*
chemical_list: D064466:Solute Carrier Family 12, Member 1
keywords: Na-K-2Cl; apical membrane; biotin ligase BirA; single molecule; trafficking