FIGURE 5:

FN recycling and fibrillogenesis require the recycling protein Rab11. (A, B) MCF10A cells either untreated (serum alone) or treated with TGF-β1/TGF-β2 (10 ng/ml for 30 min) were coimmunostained using anti-FN and either (A) anti-Arf6 or (B) anti-Rab11 and imaged using confocal microscopy. Scale bar, 5 µm (main image), 1 µm (inset). Percentage colocalization from A and B was determined using the Coloc2 plug-in in ImageJ (Materials and Methods) from single z-plane between FN and either Arf6 or Rab11 in untreated compared with TGF-β1– or TGF-β2–treated cells. Asterisk indicates significant differences in percentage colocalization (N > 15) between untreated compared with TGF-β1– or TGF-β2–treated cells (*p < 0.001, t test). Images are representative of at least three independent experiments. (C) Recycling assay as described in Figure 4F in the presence of Rh-FN in pEGFP and Rab11 (S25N)-GFP–transfected MCF10A cells. Cells either untreated (UN) or treated with 10 ng/ml TGF-β1 for 30 min were imaged. Only constructs expressing EGFP or Rab11(S25N)-GFP (as monitored by GFP expression) were imaged, and representative images are presented showing abrogated recycling of Rh-FN in Rab11 (S25N) transfectants. Arrowheads indicate fibrils. Scale bar, 2 µm. (D) MCF10A cells were transfected with pEGFP or Rab11 (S25N)-GFP as in C, immunostained using anti-FN after treatments with TGF-β1 or TGF-β2 (10 ng/ml for 30 min), and imaged using TIRF microscopy (penetration depth, 110 nm). Scale bar, 5 µm. (E) Average fibril number/cell analyzed as described in Materials and Methods from TIRF images in GFP and Rab11 (S25N) mutants in untreated (UN) cells or cells treated with TGF-β1 or TGF-β2. Fibril lengths were tracked using the NeuronJ plug-in on ImageJ. Asterisk indicates significant differences (N = 10 [ANOVA]) in fibril number between untreated GFP transfectants for untreated (UN) cells and cells treated with TGF-β1 or TGF-β2 (*p < 0.001).