Fig. 1

Endoplasmic reticulum (ER)-to-Golgi cargo transport induces transient lysosome repositioning. a HeLa cells were microinjected in the nucleus to deliver a DNA construct encoding LAMP1-GFP (green fluorescent protein) for its synchronized expression and transport from the ER. Representative images show LAMP1-GFP localization in the ER, Golgi or post-Golgi at the indicated time after microinjection. Cells were stained with DeepRed-Lysotracker, and its radial integrated fluorescence intensity was used to quantify the distribution of lysosomes as described in Methods. The graph shows the percentage of cells depicting cytoplasmically spread or perinuclear lysosome distribution (n = 30 cells). Scale bar, 10 µm. b HeLa cells expressing the human growth hormone fused in tandem to GFP and the polymerization/depolymerization FM domain (hGH-GFP-FM) were subjected to the ER-to-Golgi cargo transport assay. Lysosomes were also stained with DeepRed-Lysotracker. Images were acquired by dual time-lapse to track cargo transport (inset) and lysosome localization before and at the indicated time after addition of D/D solubilizer. The perinuclear distribution of lysosomes was quantified as in a and the values are indicated at the bottom of each image (n = 10 cells). Scale bar, 10 µm. c HeLa cells were examined by electron microscopy when hGH-GFP-FM was localized either at the ER or the Golgi complex. The ratio of lysosomes (red arrowheads) with respect to Golgi stacks (green Gs) was calculated as described in Methods (n = 3 independent experiments), and the values are indicated at the bottom of each image (ER = 1.91 ± 0.61; Golgi = 3.58 ± 0.52***). Scale bar, 500 nm. Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t tests). All t tests were conducted comparing to control cells