Figure 6

Expression of cathepsin B-eGFP and cathepsin B-C29A-eGFP chimeras in normal thyroid epithelial cells. Single channel fluorescence (A and B, left panels in D), merged (D) and corresponding phase contrast micrographs (insets in A and B, left panel, bottom, in D) taken with the confocal laser scanning microscope of FRT cells after transfection with plasmids coding for eGFP-tagged active (A) and inactive cathepsin B (B and D) as indicated. The expression pattern of both chimeric proteins was vesicular in FRT cells (arrows in A and B, green signals in D). The endogenous levels of mature cathepsins B, L and S (SC – single chain, HC – heavy chain) were estimated through immunoblotting and densitometry analysis and the cathepsins B:L:S ratio was shown to be 7:3:0 (C). The cathepsin levels were normalized against β-tubulin (C, arrowhead) that was used as a control for equal loading. The quenched activity based probe GB117 became fluorescent upon reaction with active cysteine peptidases and exhibited a vesicular staining indicative of active cysteine cathepsins in endo-lysosomal compartments (red signals in D). Note that the inactive cathepsin B-C29A-eGFP protein was localized within the same (yellow signals) or different vesicles (green signals) of normal rat thyroid epithelial cells (D), ruling out the existence of sorting signals intrinsic in cathepsin B’s primary structure. N denotes nuclei. Scale bars represent 20 µm.