Figure 5

CLIC4 interacts with MMP14 and is required for LE luminal expression of MMP14. (A) ARPE19 cell lysates and the immunoprecipitants pulled down by anti-MMP14 or control mouse antibody were analyzed by immunoblotting with the indicated rabbit antibodies. (B) The lysates of 293 T cells transfected with Flag-CLIC4 and MMP14-mCherry (left) and the immunoprecipitants pulled down by anti-Flag or control antibody (right) were immunoblotted with the indicated antibodies. Asterisks point to the MMP14-mCherry specifically co-immunoprecipitated with CLIC4. (C) Live snapshot image of co-transfected GFP-CLIC4 and MMP14-mCherry in ARPE19 cells. The low magnification photograph of a single cell is shown in the bottom right panel; the dashed lines mark the cell border. The boxed area is magnified to highlight the overlapping GFP-CLIC4 and MMP14-mCherry signals. (D) A still image of live ARPE19 cells transfected with mCherry-CLIC4 (red) and Lamp1-GFP (green). The low-magnification images taken from a single cell and the enlarged views of the boxed area (insets) are shown. (E) A snapshot of live images shown in Supplementary Movie 1. Fluorescein-dextran was added to the culture medium of ARPE19 cells transiently transfected with mCherry-CLIC4. After washing and a 6-hour chase, the cells were imaged in recording buffer. (F) Immunoblots of Dox treated (+) or untreated (−) ARPE19 cells stably expressing inducible CLIC4-sh1 plasmid (no fluorescent reporter) probed with the indicated antibodies are shown. (G) The ARPE19 cell line described in (F) was treated with (i.e., CLIC4 KD) or without (CLIC4 WT) Dox for 3 days. These cells were then transfected with MMP14-mCherry, GFP-CD63, and Flag-Rab5 Q79L for 1 day in the same Dox conditions before fixation and staining. Representative super-resolution confocal images of mCherry and GFP stained cells are shown. Arrowheads and arrows point to LE luminal and LE limiting membrane signals of MMP14, respectively. (H) Quantification of targeting of MMP14 in (G). For quantification, a threshold was adjusted to exclusively reveal the limiting membrane signal of CD63. In 120 randomly selected LEs, the ratio of the MMP14 signal within the LE lumens was deducted by subtracting its intensity on limiting membrane from total intensity on the entire LE globe. n = 120 endosomes from 24 cells. Scale bars (in C–E,G) = 10 µm.