Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - EGF is normally transported to EEA1 positive structures. HeLa cells expressing GFP-p50 and control cells were pre-treated as described in Figure 2 and were labeled with an antibody against EEA1 (blue). Confocal images are shown. High magnification views are shown in the rectangles. Bars represent 10 µm.
• Supplemental Figure 2 - EGF is transported to peripherally mis-localized LBPA positive structures. HeLa cells expressing GFP-p50 and control cells were pre-treated as described in Figure 2 and were labeled with an antibody against LBPA (blue). Confocal images are shown. High magnification views are shown in the rectangles. Bars represent 10 µm.
• Supplemental Figure 3 - GFP Rab7da expressing cells show a delayed transport of EGF from the EEA1 to the LAMP1 positive structures (A) HeLa cells expressing GFP-Rab7da and control cells were treated as described in Figure 6. The additional time points (0'+0', 10'+0', 10'+120') are shown. Cells were labeled with an antibody against EEA1 (blue). Confocal images are shown. High magnification views are shown in the rectangles. Bars represent 10 µm. (B) HeLa cells expressing GFP-Rab7da were treated as described in Figure 6. The additional time points (0'+0', 10'+0', 10'+10') are shown. Cells were labeled with an antibody against LAMP1 (blue). Confocal images are shown. Bars represent 10 µm.
• Supplemental Figure 4 - Ultrastructure of cryofixed and freeze-substituted HeLa cells after over-expression of (A-C) HA-p50 or (D-E) GFP-Rab7da versus (F) non-transfected Hela control cells; scale bar represents 500nm for all figures.
  The huge size and structural characteristics of late endosomes/lysosomes in transfected cells (as observed in immuno-labelled cryosections, see Figures 10AB and Supplementary Figure 6 A-C) allowed a tentative identification of these compartments also in the here shown high-pressure frozen, resin embedded cells. (Note that these specimens could, by technical reasons, not be immuno-labelled.) Remarkably enlarged late endosomal compartments of varying morphology are regularly seen after over-expression of (A-C) p50-HA or (D-E) GFP-Rab7da but not in (F) controls cells. The maximal diameter of these altered late endosomes/lysosomes was ca. 3µm versus ca. 0.5µm in controls. They appeared to be spherical to ovoid, or in the case of Rab7da-overexpression sometimes pleiomorphic, with more or less prominent vesicular, multi-lamellar or disorganised contents. Other endosomal compartments such as early endosomes, MVBs and multi-lamellar (dense-core) lysosomes displayed completely normal ultrastructure in any case. Similarly, all the other organelles proved morphologically unchanged in transfected cells as compared to control cells (with the possible exception of the Golgi stacks in p50 expressing cells (Burkhardt et al., 1997).
• Supplemental Figure 5 - Immuno-EM of non-transfected cells from over-expression experiments with (A) GFP-Rab7da (see Figure 10B) and (B) HA-p50 (see Figure 10A) served as controls for EGFR-labelling. No EGFR-labelling is seen in LAMP1-positive late endosomal/lysosomal compartments after 120 min of EGF stimulation (LAMP1=10 nm gold); scale bar represents 100 nm.
• Supplemental Figure 6 - Immuno-EM of GFP-Rab7da and GFP-p50 over-expressing HeLa cells after 120 min of EGF stimulation. (A) GFP-Rab7da over-expressing cells show EGFR within Rab7da-positive late endosomes (EGFR = 5 nm gold, marked by arrows; GFP = 20 nm gold), scale bar represents 100 nm. (B) Cluster of enlarged late endosomes with GFP-Rab7da immuno-labelling (GFP=20 nm gold), scale bar represents 100 nm. (C) GFP-p50 over-expressing cells show co-localisation of EGFR (5 nm gold: arrows) and LAMP1 (10 nm gold) within late endosomal/lysosomal compartments, scale bar represents 100 nm.