Figure 2.

Dx and Su(dx) Induce Notch Endosomal Trafficking with Distinct Temperature Sensitivities

(A and B) Dx coexpression promotes N endocytosis.

(C–E) N endocytosis resulting from coexpression with Su(dx) increases with temperature.

(F) Quantification of temperature dependence of N endocytosis. Localization was scored after indicated chase times as mostly plasma membrane (PM), plasma membrane and vesicular (PM = V), and mostly vesicular (V). Basal N endocytosis and Su(dx)-induced endocytosis increase with temperature. Dx-induced N endocytosis is markedly less sensitive to temperature, as is N endocytosis when Dx and Su(dx) are coexpressed.

(G and H) Endocytosed N (purple) colocalization with Rab5-GFP (green) indicated by arrowheads after coexpression with Dx (G) and Su(dx) (H).

(I and J) Time course of N progression through Su(dx) and Dx-induced N endocytic pathways.

(K) N localizes to the edge of Rab7-GFP-marked vesicle when coexpressed with Dx.

(L and M) N in Su(dx)-expressing cells (L) or Su(dx) + Dx-expressing cells (M) is localized within Rab-7-GFP-marked vesicles.

(N) Su(dx)V5 induces N endocytosis, but N is localized to Rab7-GFP-marked vesicle limiting membrane.

(O) Distance between Rab7-GFP and peak N localization in late endosomes. Increased value represents increased internalization within Rab7-GFP-marked vesicle. Su(dx) expression or increased temperature overcomes ability of Dx to retain N to the edge of the Rab7-marked limiting membrane. Su(dx)V5 prevents N transfer from the late endosomal limiting membrane. At 18°C, Su(dx) is less effective than at 25°C at transferring N into late endosome lumen.

(P) Su(dx) promotes temperature-dependent ubiquitination of Notch, but Su(dx)V5 has no ubiquitination activity.

(Q) Su(dx)V5 increases N signaling independently of temperature.

Data in (F), (I), (J), (O), and (Q) are displayed as means ± SEM (n = 3, minimum 60 cells or vesicles scored per repeat), p < 0.05 for all differences stated in legend (Student t test). See also Figure S2.