Fig. 4.

(A–G) Endosomal accumulation of PH-GFP (PI(4,5)P2) was increased in cnt-1(tm2313) and rab-10(q373) mutants but was decreased in arf-6(tm1447) mutants. Representative confocal images are shown for PH-GFP in wild-type animals (A and E) and in cnt-1(tm2313) (B), arf-6(tm1447) (C), and rab-10(q373) (F) mutants. (Scale bar: 10 μm.) The arrowhead in A indicates basolateral membrane. (D and G) Quantification of average total intensity of PH-GFP per unit area. Error bars indicate SEM. n = 18; three different regions of the intestine (defined by a 100 × 100 pixel box positioned at random) were sampled in six animals of each genotype. ***P < 0.001, one-tailed Student’s t test. (H) PI(4,5)P2 levels in cnt-1(tm2313), arf-6(tm1447), and rab-10(q373) mutants and in cnt-1(tm2313);arf-6(tm1447), rab-10(q373);arf-6(tm1447) double mutants. Whole-animal lipid extracts were prepared using TLC and were analyzed by gas chromatography. PI(4,5)P2 measurements were normalized to the abundant phospholipids (phosphatidylcholine and phosphatidylinositol) in each sample. Membrane PI(4,5)P2 levels are higher in cnt-1 and rab-10 mutants than in wild-type animals. Notably, consistent with the quantification of subcellular PH-GFP shown in G, the PI(4,5)P2 level is substantially higher in rab-10 mutants than in wild-type animals. Conversely, the PI(4,5)P2 level is moderately decreased (35%) in arf-6 mutants as compared with wild-type animals. The PI(4,5)P2 level appears fairly normal In cnt-1(tm2313);arf-6(tm1447) double mutants but is twofold higher in rab-10(q373);arf-6(tm1447) double mutants than in wild-type animals. The loss of ARF-6 reduces the elevated PI(4,5)P2 levels in rab-10 mutants by about 40%. Data shown are the mean values and SEM from three independent experiments.