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Fig. 1. Translocation of Dd-TRAP1 from the cell cortex to mitochondria is induced by prestarvation factor(s). (A) Change in subcellular localization of Dd-TRAP1 due to increased cell density. Ax-2 cells grown at the indicated cell density were fixed and immunostained with the Dd-TRAP1 antibody. Dd-TRAP1 is translocated from the cell cortex to cytoplasmic granules as the density of growth-phase cells increases. Bar, 5 µm. (B) Ax-2 cells grown at 4x10⁶ cells/ml were incubated with 1 µM MitoTracker for 30 minutes and fixed for immunostaining with the Dd-TRAP1 antibody. The merged image indicates that Dd-TRAP1 is localized in mitochondria at a high cell density. Bar, 5 µm. (C) Effect of CGM (conditioned growth medium) on the Dd-TRAP1 translocation. CGM was prepared from Ax-2 cells that had been grown to a high cell density (1.5x10⁷ cells/ml). Cells grown at a low cell density (3-5x10⁵ cells/ml) were allowed to adhere to coverslips, and incubated in the CGM. After the indicated time of incubation, the cells were fixed and immunostained with the Dd-TRAP1 antibody. Bar, 5 µm. (D) Cells grown at high cell density (8x10⁶ cells/ml) were harvested and transferred to fresh growth medium at low cell density (5x10⁵ cells/ml). After 30 minutes of incubation, cells were fixed and immunostained with the Dd-TRAP1 antibody. Compared with cells before transfer to CGM (a), it is evident that Dd-TRAP1 goes back to the cell cortex after 30 minutes of incubation in fresh growth medium (b). Bar, 5 µm.

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