Fig. 1. Preconditioning induces the translocation of endosomal-lysosomal organelles to the extreme periphery of the cell. Hepatocytes were incubated under control conditions prior to (control) or after being preconditioned by a cycle of hypoxia-reoxygenation (A) or by a 15 minute incubation with 1 µM CGS21680 (B). Cells were allowed to attach to glass coverslips, fixed and permeabilized, and processed for immunofluorescence confocal microscopy. Endosomes and lysosomes were identified by immunofluorescent detection of CD or Lamp-1. Representative images are shown. (A) Hypoxic preconditioning (PC) caused the dislocation of endosomes and lysosomes from the perinuclear region (see controls, Co) toward the cellular periphery. (B) CGS21680-preconditioned hepatocytes were stained for both CD (red fluorescence, panel b) and for actin (green fluorescence, panel c). Staining of cortical actin marked the cell border. Cell morphology can be appreciated in the phase contrast image (panel a). The translocation of endosomes and lysosomes to the extreme periphery of the cells can be appreciated in panel d, showing the overlap of actin and CD staining. The arrow in panel d points to the plasma membrane region in which cortical actin appears disassembled, as expected in the exocytosis process (Miyake et al., 2001), while lysosomal CD appears to be extruded from the cell (see also panels b and c). (C) Typical cytofluorograms of cell surface expression of Lamp-1 are shown. The positivity for this lysosomal membrane protein is increased in hepatocytes preconditioned by transient hypoxia (PC) or by CGS21680 treatment (CGS).