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. 2014 May 22;157(5):1160–1174. doi: 10.1016/j.cell.2014.03.050

Figure 3.

Figure 3

Su(dx) and Dx Regulate Notch Trafficking by Distinct Endocytic Routes

(A) When coexpressed with Su(dx), endocytic N (purple) extensively colocalizes with GPI-GFP (green, arrowheads).

(B) Enlarged region boxed in (A) showing additional colocalization of Su(dx) (blue, arrowhead).

(C) When coexpressed with Dx, endocytosed N does not colocalize with GPI-GFP.

(D) Combined expression of Su(dx) and Dx partially relocalizes N into GPI-GFP-marked vesicles (arrowheads).

(E) N-positive vesicles were scored as percent GPI-GFP positive or negative.

(F) Dx-induced N endocytosis is unaffected in S2 cells treated with mβCD to deplete cholesterol.

(G) Su(dx)-induced N endocytosis is suppressed in cholesterol-depleted cells.

(H) N localization scored as mostly plasma membrane (PM), plasma membrane and vesicular (PM = V), and mostly vesicular (V) at different cholesterol levels. Cholesterol depletion suppresses Su(dx)-induced N endocytosis, and this is rescued by reloading cells with cholesterol. Dx-induced N endocytosis is unaffected by cholesterol depletion. Overloading of cells with cholesterol induces N endocytosis.

(I) Cholesterol overload promotes N endocytosis into GPI-positive vesicles even when Dx is expressed.

(J–M) In (J), N (purple) does not colocalize in wing imaginal disc epithelial cells with GPI-GFP (green) when Dx is expressed but does colocalize with GPI-GFP (arrowheads) following expression of Su(dx) (K) or Su(dx)-ΔHECT (L) or after coexpression of Su(dx) with Dx (M).

(N and O) Time course of endocytosed N and GPI-GFP localization. (N) In S2 cells, Dx-induced N endocytosis is predominantly through GPI-GFP negative vesicles, while Su(dx) drives N endocytosis through GFP-GFP-positive compartments. (O) Temperature increases the localization of Notch to GPI-GFP-positive compartments when Su(dx) and Dx are coexpressed. An increased proportion of N and GPI-GFP colocalization is observed after longer chase periods.

(P) N (blue), endocytosed after Dx expression is localized (arrow) in an endocytic vesicle, marked with Dextran (red) immediately adjacent to a GFP-GPI-marked compartment.

Data in (E), (H), (I), (N), and (O) are displayed as means ± SEM (n = 3, minimum of 60 vesicles or cells scored per repeat), p < 0.05 for all differences stated in legend. See also Figure S3.