Figure 4
APP is processed in the lysosome by a γ-secretase like activity. SN56 cells were transiently transfected with βAPP-paGFP, GalT-CFP, and LAMP1-mRFP. Cells were alternately photoactivated with 405 nm light and imaged in the Golgi for 15 minutes, and then imaged every 30 seconds for 1 hour. a) Shows the accumulation of photoactivated APP-paGFP in the lysosome after 15 minutes, follow by its near complete clearance after 45 minutes. Arrowheads denote areas of colocalization. (See also Additional file 3: Video S1) b) Transiently transfected SN56 cells were pretreated with 100 μm chloroquine for 30 minutes prior to imaging. After chloroquine treatment APP is still visible in lysosomes after 45 minutes (See Additional file 6: Video S3). c) Cells treated with 0.5 μm L685, 458 (γ-secretase inhibitor) overnight prior to photoactivating/imaging. L685, 458 treatment substantially increases the accumulation of photoactivated βAPP-paGFP in lysosomes, and substantially decreases its cleance. Scale bars represent 5 μm (See Additional file 7: Video S4). d) Cleavage of βAPP-paGFP was determined by measuring the loss of FL-APP (black open triangles) and βAPP-paGFP (black closed circles) from LAMP1 labeled compartments. Values were averaged and normalized to begin at 100%. Overlaid in green squares is the loss of fluorescence of EGFP in the identical imaging protocol. Error bars represent SEM. (* = p < 0.05) (e) Shows the clearance of photoactivated APP-paGFP cells that were treated with 100 μm chloroquine for 30 minutes before imaging (n = 9) or with 0.5 μm L685, 458 (γ-secretase inhibior) (n = 9). Error bars represent SEM.