Figure 5

Visualization of active cysteine cathepsins in HTh74 cell cultures. Merged fluorescence micrographs of HTh74 cells growing in multilayers as detected after incubation with the fluorophore-conjugated activity based probe DCG-04 (green signals), counter-staining of nuclear DNA with DRAQ5™ (blue signals), and confocal laser scanning microscopy (A, B, D). The activity based probe DCG-04 binds to the active site of mature cysteine peptidases in a 1:1 ratio and visualizes proteolytically active cysteine proteases, only. Three-dimensional distribution of cysteine peptidase representative signals is sketched in C. Single optical sections taken in xy were obtained in different focal planes (A1-A3, cf. lines in C) and compiled in a zero-projection as an extended focus (B) as well as in xz-direction as a side view (D). Active cysteine peptidases were localized to variably sized vesicles throughout the cell (arrows). In addition, active cysteine peptidases were secreted from HTh74 cells and associated in aggregates with the plasma membrane (arrowheads). Note that active cysteine peptidases were secreted in a non-directional fashion at all poles of HTh74 thyroid carcinoma cells (D, arrowheads). Scale bars represent 10 µm.