Figure 1.

Endolysosomes Are the Principal Sites of Cathepsin B Catalytic Activity

Terminal endocytic compartments of NRK cells were loaded with DexOG for 4 hr followed by a 20 hr chase in DexOG-free medium.

(A) Part of a single cell showing that cresyl violet fluorescence (red) was present only in a subset of terminal endocytic organelles containing pre-loaded dextran after incubation with cathepsin B MR substrate for 1 hr. Arrowheads show examples where terminal endocytic organelles have not hydrolyzed the MR substrate.

(B) Pearson’s correlation coefficients (R(r); mean ± SEM of nine or more cells within a single experiment) for colocalization of pre-loaded DexOG and liberated cresyl violet fluorescence after hydrolysis of cathepsin B MR substrate for different times (see also Figure S1A).

(C) CLEM of the cathepsin-active organelles. DexOG-positive, MR-positive organelles were identified by confocal microscopy before processing for TEM to show their ultrastructure. The boxed region of the confocal image and corresponding section in the TEM shows the ultrastructure of four cathepsin-active endolysosomes. The enlargements show that these organelles contain multi-lamellar intralumenal membranes and electron dense content.

(D) An image from an adjacent confocal plane to that shown in (C) with enlargements and corresponding TEM image of the boxed region identifying a DexOG-positive, MR-negative terminal endocytic organelle (arrowhead) within the constellation of endolysosomes. The TEM image of this organelle reveals it to be a granular dense core lysosome (boxed region of TEM image and inset).

Scale bars represent 5 μm (A), 5 μm (C, light microscopy [LM]), 1 μm (C, TEM), 500 nm (C, TEM enlargements), 5 μm (D, LM), 1 μm (D, TEM), and 200 nm (D, TEM inset). See also Figure S1.