pIIb-specific sorting of Spdo toward late endosomes. (A–C″) Distribution of NiGFP4Cherry5 in SOPs (nlsFP670) at t = 15 (in minutes after anaphase). Two confocal sections (apical, A–A″; subapical, B–B″) and a transverse view (C–C″) are shown. GFP-positive endosomes are indicated with arrows in pIIb (B′ and C′). (A′) Note the low level of Notch in pIIa/pIIb cells. (D) Interpretation of the distribution of the SpdoCherryGFP fluorescence along the endocytic path in pIIb. The GFP fluorescence is initially strong at the plasma membrane of pIIb and then decreases over time as Spdo is internalized. As Spdo is endocytosed, the GFP fluorescence increases in both GFP-positive early/sorting endosomes (green endosomes) and Cherry-positive endosomes (orange and red endosomes). Numb is required to both inhibit the recycling of internalized Spdo and promote its sorting toward late endosomes. (E and F) Distribution of SpdoCherryGFP at t = 20 (in minutes after anaphase in wild-type and numb mutant clones [marked by the loss of nuclear GFP]). (F) Note the GFP fluorescence of SpdoCherryGFP at the plasma membrane of both daughter cells in the numb clone. (G) Box plots of the SpdoCherryGFP fluorescence intensities (GFP and Cherry, as indicated) measured in pIIb (top) and pIIa (bottom) cells in wild-type (white) and numb mutant cells (gray) at t = 10, 20, and 30 min. Total fluorescence intensities (y axis) measured in GFP-positive (early/sorting) endosomes (left) and Cherry-positive (late) endosomes (right) are shown. GFP increased significantly in the Cherry-positive endosomes of wild-type pIIb (blue asterisks indicate P < 0.05; see Results for p-values). Conversely, Cherry increased significantly in the GFP-positive endosomes of wild-type pIIb. Corresponding values did not significantly change in numb mutant cells. This indicated that GFP-positive Spdo was sorted toward late Cherry-positive endosomes in a Numb-dependent manner in pIIb. No significant changes were in wild-type pIIa. However, statistically significant changes were observed in both pIIb and pIIa in numb mutant clones (red asterisks indicate P < 0.05). These changes likely reflected the Numb-independent endocytosis of Spdo that is present at higher levels at the plasma membrane in dividing numb mutant SOPs. Box and whisker plots are uniform in their use of the box: the bottom and top of the box are the first and third quartiles, and the band inside the box is the second quartile (the median). The ends of the whiskers represent the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile (often called the Tukey box plot). Outliers are shown as small circles. Bars, 5 µm.