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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01180

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The lateral mobility of NHE3 on the apical membrane of renal epithelial OK cells is limited by the PDZ domain proteins NHERF1/2, but is dependent on an intact actin cytoskeleton as determined by FRAP

¹ Departments of Physiology and Medicine, Gastroenterology Division, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
² Department of Molecular Physiology and Biophysics and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

View larger version (21K): [in a new window]   Fig. 1. OK and OK/NHERF2 cells were selected by acid suicide to be NHE3 null. OK cells, which had been cloned to remove fibroblast contamination (Noel et al., 1996), were selected by acid suicide (see Materials and Methods). The intracellular pHi was measured with BCECF over time, following acidification by NH4Cl pulsing (Levine et al., 1993). Arrow indicates Na+ addition. AS refers to acid suicide. NHE3 null OK cells were used for transient transfection.

View larger version (114K): [in a new window]   Fig. 2. NHE3-EGFP expression in transiently transfected OK cells. Images were collected 48 hours after transfection with NHE3-EGFP, during which time the cells were incubated with serum-free media. Fluorescence micrographs were taken with a 100x objective. NHE3-EGFP localized in three pools in OK cells: in the juxtanuclear (JN) area (as previously described) (D'Souza et al., 1998; Akhter et al., 2002) (A), in the microvilli of the apical plasma membrane above the JN or in a non-JN area (B). Bar, 10 µm. (C) XZ reconstruction showing NHE3-EGFP distribution in apical membrane and JN area. (D) The three pools of NHE3-EGFP in OK cells. A and B were oversaturated on purpose to show both microvillus and intracellular NHE3 locations.

View larger version (87K): [in a new window]   Fig. 3. FRAP analysis of NHE3-EGFP in the three regions in OK cells. Fluorescence photobleaching was performed and recovery analyzed in the three regions shown in Fig. 2 – intracellular JN, apical surface above-JN and non-JN of NHE3-EGFP in OK cells. Images show prebleaching (A), immediately post bleaching (B) and after recovery (450 seconds after bleaching) (C) at the level of the apical plasma membrane. The prebleaching intensity (Fi), intensity just after bleaching (F0) and final intensity after full recovery (F) are shown in D. FRAP data were collected every 9 seconds up to 50 images. The mobile fraction (Mf) and effective diffusion coefficient (Deff) of NHE3-EGFP in the three pools in OK cells were obtained by fitting the photobleaching curve according to Materials and Methods (see E and F). n=12 cells studied for Cyto-JN, n=15 for above-JN and n=21 for non-JN. Data were from three separate experiments. P values for Mf were in comparison to cyto-JN (unpaired t-test).

View larger version (69K): [in a new window]   Fig. 4. NHE3-EGFP did not recover after photobleaching in either the apical plasma membrane or JN areas after DSS cross-linking. OK/NHE3-EGFP cells had fluorescence recovery studied after the cells were exposed to the membrane-penetrable cross-linking reagent DSS (disuccimidyl suberate) (1 mM for 1 hour at 37°C). The NHE3-EGFP fluorescence did not recover in either the apical domain or in the intracellular juxtanuclear (JN) region after photobleaching. (A) Time-course montage after photobleaching (the numbers at the bottom are seconds after the bleach) of apical membrane NHE3-EGFP in OK cells. Magnification 100x objective. (B) Quantitative fluorescence recovery of NHE3-EGFP in the apical plasma membrane. Similar results were obtained of NHE3-EGFP in the intracellular JN domain. Note, the apical membrane studied (A and B) included NHE3-EGFP over the JN plus the non-JN domain. Mobile fractions for NHE3-EGFP in Cyto-JN and the apical plasma membrane (PM) are shown in C. n=5 for Cyto-JN and n=6 for PM from two separate experiments.

View larger version (34K): [in a new window]   Fig. 5. The contribution to apical membrane NHE3-EGFP recovery after photobleaching of intracellular NHE3-EGFP was negligible when non-JN NHE3 was studied but was significant in the above-JN region. Fluorescence recovery was studied after 30 minutes exposure at 4°C to the water-soluble cross-linking reagent, BS3 (10 mM). NHE3-EGFP recovery was performed on the intracellular JN (cyto-JN) (A), apical plasma membrane above-JN (B) and non-JN (C) domains. The mobile fractions (D) and diffusion coefficients (E) of the NHE3-EGFP domains were determined by FRAP. Data shown are mean±s.e.m. * P values compared with cyto-JN are shown (unpaired t-test). n=7 for cyto-JN and non-JN; n=6 for above-JN.

View larger version (16K): [in a new window]   Fig. 6. Decreasing surface NHE3 by inhibiting phosphoinositide 3-kinase did not alter fluorescence recovery of non-JN NHE3-EGFP remaining in the apical surface. OK/NHE3-EGFP cells were incubated with 50 µM LY294002 for 30 minutes at 37°C, then fluorescence recovery measurements were performed with analysis of apical non-JN NHE3-EGFP. The result shown is a representative FRAP experiment with initial fluorescence intensity before photobleaching set at 100%. Data were collected as 50 images every 9 seconds. Mf=50.5±4.6% and Deff=(3.9±1.2) x10–10 cm2/second, for six cells in two separate studies on both untreated and LY294002-treated cells. Data shown are mean±s.e.m.

View larger version (102K): [in a new window]   Fig. 7. NHE3-EGFP, NHERF2, ezrin and F-actin colocalize in the OK/NHERF2/NHE3-EGFP cell apical membrane. OK cells transiently expressing NHE3-EGFP were plated on a glass-bottom 35 mm plastic culture dish and processed for confocal imaging. Cells were fixed and labeled either with polyclonal anti-NHERF2 (A2 and E2), monoclonal anti-ezrin (B2) or phalloidin (C1-3 and D1). Secondary antibodies were either Alexa-568-conjugated goat anti-rabbit IgG for NHERF2 staining or Alexa-595-conjugated goat anti-mouse IgG for ezrin staining. The green channel shows the distribution of NHE3-EGFP and red channels show NHERF2 (A2, E2), ezrin (B2) and F-actin (C1-3, D1). Overlayed images are shown in the right column. XZ sections show colocalization of NHE3-EGFP with NHERF2 (A3), ezrin (B3) and F-actin (D3) in microvilli. C1-3 shows F-actin staining at the basal stress fibers (C1), the lateral cell surface (C2) and apical membrane microvilli (C3) in polarized OK cells. Images D1-D3 show F-actin (D1) and NHE3585-EGFP (D2) staining at the apical plasma membrane. D3 is a composite image between D1 and D2. F-actin colocalized with NHE3585-EGFP only at the microvilli (D3a) but not in the subapical juxta nuclear region (D3b). XZ section images E1-E3 show NHE3585-EGFP (E1) and NHERF2 (E2). E3 is a composite image between E1 and E2. NHERF2 did not colocalize with NHE3585-EGFP at the microvilli. However, NHERF1 still shows normal microvilli distribution at the microvilli (F2) and colocalized with microvillus NHE3585-EGFP (F3). These OK cells endogenously expressed ezrin and NHERF1, stably expressed NHERF2 and transiently expressed NHE3-EGFP. Images were taken with a 100x objective. Bars, 10 µm.

View larger version (42K): [in a new window]   Fig. 8. Effect of PDZ domain proteins NHERF1/2 on the apical surface lateral mobility of non-JN NHE3-EGFP. Photobleaching experiments were done on OK/NHE3-EGFP (A), OK/NHERF2/NHE3-EGFP (B) and OK/NHERF2/NHE3585-EGFP cells (C). Initial fluorescence was set to 100%. The estimated mobile fractions and effective diffusion coefficients are shown in (D) and (E). P values are in comparison with OK/NHE3-EGFP (unpaired t-test). n=21 for OK/NHE3-EGFP, n=11 for OK/NHERF2/NHE3-EGFP and n=9 for OK/NHERF2/NHE3585-EGFP cells from at least three separate experiments. NS, not significant.

View larger version (75K): [in a new window]   Fig. 9. Efects of latrunculin B on the mobility of NHE3-EGFP and NHE3585-EGFP. OK cells transfected with NHE3-EGFP or OK/NHERF2 cells transfected with NHE3585-EGFP were incubated with latrunculin B (0.1 or 0.05 µM) at 37°C for 30 minutes. Latrunculin B (1 µM, 30 minutes) completely abolished the presence of microvilli (data not shown). F-actin distribution at the apical microvilli was partially disrupted with 0.1 µM latrunculin B for 30 minutes (A). In most of the cells, the apical NHE3-EGFP moved intracellularly as shown in (B). Cell exposed to latrunculin B (0.05 µM for 30 minutes) still had microvilli and NHE3-EGFP remained in the microvilli (C). OK/NHE3-EGFP cells exposed to latrunculin B (0.05 µM) for 30 minutes at 37°C had fluorescence recovery analyzed in the apical plasma membrane non-JN and in the intracellular JN domains. There was no change in Mf or Deff of NHE3-EGFP in the intracellular JN domain (Mf=37.0±8.3% and Deff=(3.9±1.3) x10–10 cm2/second; n=6). Apical non-JN NHE3-EGFP had decreased Mf compared with cells not treated with latrunculin B (D, left). Similar studies of apical surface non-JN NHE3585-EGFP in OK/NHERF2/NHE3585-EGFP cells also showed a large decrease in mobile fraction (Mf) caused by this latrunculin B treatment (D, right). The effective diffusion coefficients with and without latrunculin B (0.05 µM) are shown in (E) and did not significantly change with latrunculin B. Data are mean±s.e.m. 7 cells studied for latrunculin B treatment in D and E.

View larger version (49K): [in a new window]   Fig. 10. Mf of OK/YFP-GL-GPI is not dependent on NHERF2 expression and is not altered by latrunculin B. FRAP study was performed with OK and OK/NHERF2 cells transiently transfected with YFP-GL-GPI. (A) The YFP-GL-GPI was present in microvilli in both OK/NHERF2/YFP-GL-GPI and OK/YFP-GL-GPI (not shown) cells. (B) The mobile fractions of microvillus YFP-GL-GPI were estimated in OK/YFP-GL-GPI, OK/NHERF2/YFP-GL-GPI and OK/NHERF2/YFP-GL-GPI + latrunculin B(0.05 µM, 30 minutes) as described above. There was no significant change in Mf of YFP-GL-GPI in OK cells stably expressing NHERF2 and latrunculin B-treated cells. EGFP settings of photobleach, excitation (488 nm) and emission (515 nm) were used for the YFP studies.

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