Fig. 6. SKD1(E235Q)-expression induces the accumulation of hybrid organelles. GFP-SKD1(E235Q)-expressing NRK cells are indicated by asterisks (A-D) and the localization of GFP-SKD1(E235Q) is directly visualized with GFP (green, A-D). (A) TR-dextran-labeled lysosomes were enlarged in the cells expressing GFP-SKD1(E235Q). (B,C) The enlarged lysosomes contained cathepsin L, a lysosomal enzyme and LBPA, a marker for the late endosomes. (D) Triple labeling of the cells expressing GFP-SKD1(E235Q) by GFP (green), the preloading TR-dex (red) and the mAb against lgp120 (blue) was visualized through a confocal microscope and revealed the heterogeneity of E235Q compartments (asterisks). Purple arrowheads reveal the co-localization of lgp120 and TR-dex in the enlarged hybrid organelle. Light blue arrows indicate the co-localization of lgp120 and GFP-SKD1(E235Q) in the compartments that lack TR-dex and thus reveal that they are E235Q compartments derived from either late endosomes or a subset of endosomes that accumulate endocytosed lgp120. The aberrant structures only labeled with GFP-SKD1(E235Q) represent E235Q compartments that are separated from late endocytic compartments. (E) The number and diameter of TR-dex-positive structures were measured in three separate experiments (in total nine infected cells and 16 uninfected cells). The size distribution (vesicle diameter, µm) of the TR-dex-positive structures is represented as a histogram of the mean vesicle number/cell. The majority of TR-dex-positive structures ranged between 0.2 and 0.8 µm in diameter in uninfected cells, while the size range of infected cells shifted between 0.8 and 1.4 µm. Bar, 20 µm.

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