Figure 7.

Cripto recruits Nodal to nonclathrin, noncaveolar endocytic compartments marked by Flotillin-1 and -2. (A) Antibody uptake by Flag-tagged Nodal (red) in early endosomes expressing TfR was undetectable after 5 min irrespective of the presence or absence of Cripto. (B) Nodal uptake in membranes marked by GFP–Caveolin-1 in the absence and presence of Cripto. (C) Cripto mediates Nodal uptake in membranes expressing GFP–Flotillin-1. (D) Quantification of colocalization of internalized Nodal with GFP–Caveolin-1 and GFP–Flotillin-1 (^***P<0.001, Student's t-test). (E) Immunogold-labelled anti-Flag antibody internalized by Flag–Nodal in cells transfected with (bottom) or without Cripto (top) decorates uncoated membrane invaginations (arrows). When co-transfected with Cripto, Nodal was also detected at the surface (arrowhead). (F) Immunofluorescence staining of antibody that was internalized by Nodal in transfected COS1 cells (left panel). In cells treated with nystatin (1 h), antibody is not internalized, but instead stains the cell membrane (red arrowheads, right panel). (G) Anti-Cripto western blot of transfected COS1 treated for 4 h with or without nystatin. γ-Tubulin was stained as a loading control. (H) Western blot analysis of whole lysates and conditioned medium of 293T cells that were co-transfected with Cripto and the proteins indicated. Inhibition of palmitoylated proteins by 2-bromo-palmitate (2BP) reduced Nodal secretion and dramatically inhibited precursor processing by endogenous PCs and Flag-tagged Furin (lanes 1, 2, 4 and 5), and to a lesser extent exogenous Flag–PACE4 (lane 6). (I) Incubation of cells with 2-BP inhibits both Nodal signalling in a luciferase reporter assay (left panel) and Nodal-mediated antibody uptake (right panels).