Figure 1.

Nhe2/NHE3 activity is required for Fz-mediated Dsh recruitment. (a) Genome-wide RNAi library screen for Fz-mediated Dsh recruitment in D. melanogaster S2R+ cells. The cells were plated in 57 384-well plates containing dsRNA against 90% of D. melanogaster genes, transfected with myc–fz and dsh–GFP plasmids and analysed visually (see Methods). (b, c) Screen hits were defined by a × 20 microscope field with >10 cells showing defective Dsh–GFP recruitment. Control (Relish dsRNA) showed complete recruitment (b, see enlarged cell, inset), Nhe2 dsRNA caused defects in recruitment (c), see arrowheads and inset for defects. (d, e) HEK293T cells expressing Myc–Fz and Dsh–GFP were treated with the NHE3 inhibitor S3226 at 50 µM for 12 h. Treatment led to a redistribution of Dsh–GFP (e) as opposed to the control (DMSO) (d). (f) NHE3 knockdown caused defective Dsh recruitment (64.3% ± 2.3%, compared with 31.6% ± 4.3% in controls, this baseline defect in Dsh recruitment is due to heterogenous Fz and Dsh expression in these cells). Treatment with 30 mM NH₄Cl to increase pH_i (mean ± s.e.m. from three independent experiments, measured in f, right graph) rescued the recruitment defect in NHE3-knockdown cells (mean ± s.d., n= three independent experiments, *P < 0.001, measured in f left graph). (g–i) Alkalinization was transient with gradual pH normalization within 20 min. Images for all three conditions in f are shown. (j–p) Intracellular acidification interferes with Dsh recruitment. S2R+ cells were prepulsed for 30 min with 30 mM NH₄Cl and then switched to a Na⁺-free medium for 12–16 h. This treatment caused a stable reduction of pH_i to 7.1 (measured by E²GFP expression²⁵ and pH calibration), resulting in defective Dsh recruitment (k–m). Incubation in a Na⁺-free medium without prepulsing (pH_i 7.28) or in Na⁺-containing medium after prepulsing (mean ± s.d., n = 4 independent experiments, *P < 0.001) mildly affected recruitment. For quantification (j), only cells expressing Fz at their cell surface were counted. Note robust recruitment in untreated cells with pH_i 7.45. (n–p) In a second assay, pH_i was lowered by applying K⁺/nigericin for 4 h and clamping pH_i according to an extracellular buffer (pH 6.8). This did not affect membrane association of the phosphatidylserine-binding LactC2–RFP but did affect Dsh membrane localization. Scale bars represent 10 µm.