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.

Vesicle-associated Membrane Protein 3 (VAMP3) Mediates Constitutive Trafficking of the Renal Co-transporter NKCC2 in Thick Ascending Limbs

“…Steady-state surface NKCC2 expression is maintained by a balance between constitutive exocytic delivery, endocytic retrieval, and recycling ( 2 – 6 , 9 ) that occurs in the absence of stimuli. To test whether VAMP3 mediates constitutive NKCC2 exocytic delivery, we silenced VAMP3 via shRNAs and measured NKCC2 exocytic delivery.…”

“…Under baseline conditions (absence of external stimuli), NKCC2 is present at the apical surface and also in intracellular vesicles in the subapical space ( 1 ). NKCC2 undergoes constitutive exocytic delivery ( 2 , 3 ), endocytic retrieval, and recycling ( 4 – 6 ) in the absence of any stimulation, maintaining steady-state surface NKCC2 expression at a baseline level. This process of NKCC2 trafficking is a critical determinant of the absorptive capability of the TAL ( 7 – 9 ).…”

Thick Ascending Limb Sodium Transport in the Pathogenesis of Hypertension

“…However, even though the number of NKCC2 proteins in the apical membrane may be constant, there is continuous insertion into and retrieval of transporters. The retrieval rate for NKCC2 measured by total internal reflection fluorescence microscopy in thick ascending limb cells is~1%/min (24,320).…”

Limited dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction

“…We therefore utilized TIRF to image Dvl2-GFP near the membrane to achieve high spatiotemporal resolution with high signal-to-noise ratio. To avoid issues of ligand accessibility we developed an apical TIRF imaging setup (Jaykumar et al, 2016) in which the cells grown on PET filters are inverted with their apical sides down, with or without Wnt3A stimulation (Figure 7a,b). In both cases we observed many diffraction-limited low intensity spots per cell, indicative of complexes containing small numbers of GFPs.…”