Figure 5. Bmper and Bmp4 are shuttled to the lysosome through endocytic trafﬁcking. Bmper locates near a caveolin compartment at the cell surface and internalizes into early endosomes. (a) MECs cultured at 4°C and treated with Bmper (6 nM) and Bmp4 (0.6 nM) were analyzed by ﬂuorescent immunoconfocal mi- croscopy. A signiﬁcant portion of the Bmper protein (green) appears to be localized near a membrane surface caveolin (red) compartment (arrow) and throughout ﬁlopodia. (b) After a 4°C incubation, MECs treated with Bmper (6 nM) and Bmp4 (0.6 nM) were permitted to internalize at 37°C. Bmper (green) is detected in a fraction (yellow) of the early endosomes expressing the marker EEA1 (red). (c) The same immunoconfocal analysis was used to charac- terize late endocytic to lysosomal translocation using the marker Rab7 (bottom, red) to co- localize with Bmper (green) and Bmp4 (red). MECs were treated with 0.6 nM Bmp4 and 6 nM Bmper for 1.5 h before ﬁxing. Signiﬁcant colocalization of Bmper and Bmp4 overlaps with Bmper and Rab7 colocalization (right panel, yellow). counterparts (unpublished data). To explore further the pathway of Bmper and Bmp4 internalization, we performed transmission electron microscopy at selective time points (Fig. 4 b) using 25-nm immunoparticles to detect Bmper and 5-nm particles to detect Bmp4. At early time points, Bmp4 and Bmper congregated along the cell membrane (Fig. 4 b, i), presumably as they associate together with Bmp receptors. The Bmp4–Bmper heterodimers soon coalesced over a dense membrane that is characteristic of clathrin-dependent invaginations (Fig. 4 b, ii and iii). These clathrin- coated pits were subsequently visualized within the cytoplasm (Fig. 4 b, iv), and transfer of these components to mature endo- somal compartments was apparent (Fig. 4 b, v, arrow). In the ab- sence of Bmper, Bmp4 was not detected intracellularly by electron microscopy after 1.5 h of treatment. Collectively, these data are consistent with a clathrin-dependent mechanism for internalizing both Bmper and Bmp4, which is consistent with recent papers describing clathrin-dependent internalization of TGF- and Bmp receptors (Di Guglielmo et al., 2003; Hartung et al., 2006). Bmper and Bmp4 undergo endocytic trafﬁcking to the lysosome We used fluorescence confocal microscopy to further character- ize the pathway by which Bmper and Bmp4 traffic (Fig. 5). MECs treated with Bmper and Bmp4 were first incubated at 4°C to capture the cell surface localization of Bmper. Bmper was located in a filamentous pattern at the cell surface, as well as in concentrated areas (possibly lipid rafts) where early endo- somes begin to traffic within the cell and near filopodia (Fig. 5 a). After internalization (1-h treatment at 37°C), Bmper and Bmp4 were detected in a fraction of vesicles expressing the marker EEA1, but were not detected in caveolin-1– or phosphorylated caveolin-1–containing vesicles below the cell surface (Fig. 5 b and not depicted). This pattern is remarkably similar to reports describing cell surface localization of receptor tyrosine kinases in lipid raft compartments followed by clathrin-dependent inter- nalization into an endocytic transport pathway (Mukherjee et al., 2006). We explored the colocalization of Bmper and Bmp4 602 JCB • VOLUME 184 • NUMBER 4 • 2009