Termination of autophagy and reformation of lysosomes regulated by mTOR

Abstract

Autophagy is an evolutionarily conserved process by which cytoplasmic proteins and organelles are catabolized. During starvation, the protein TOR (target of rapamycin), a nutrient-responsive kinase, is inhibited, and this induces autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes, which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of the autophagosome cargo in autolysosomes, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly understood. Here we show that mTOR signalling in rat kidney cells is inhibited during initiation of autophagy, but reactivated by prolonged starvation. Reactivation of mTOR is autophagy-dependent and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell-a process we identify in multiple animal species. Thus, an evolutionarily conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.

Figure 1

Lysosome homeostasis during starvation. (a) LAMP1⁺ lysosome size and quantity in NRK cells (dotted outline) starved for hours (h). Error bar show s.e.m (n=3). (b) Starved NRK cells expressing LAMP1-YFP show tubules (arrows). Box is expanded at right. (c) Immuno-TEM (gold marks LAMP1) of starved NRK cells. Continuity (arrow) illustrated between autolysosome (red star) and tubule. (d) LAMP1-YFP expressing NRK-LC3 cells as in (a). Arrows show reformation tubules. (e) 3-dimensional reconstruction of a cell from (d). (f) Time-lapse images (seconds) of starved LAMP1-YFP-expressing NRK cells. (g) Time-lapse images (minutes) of starved LAMP1-PAGFP or LAMP1-RFP-expressing NRK cells after photo-activation. Scale bars = 5 um.

Figure 2

Proto-lysosome formation and maturation. (a) - (c) Starved LAMP1-YFP-NRK cells stained with Lysotracker or DQ-BSA. (c, right) TEM of purified lysosomes. Magnification (4x) (d), immunofluorescence (e) or immuno-EM (f) using LAMP1 antibody of (c). (g) Density gradient fractions of NRK cells starved for hours (h), blotted for LAMP2 or Cathepsin D. 8h fractions 7 and 9 analyzed by TEM (center). Fraction 10 = gradient top. (h) Starved LAMP1-PAGFP NRK cells were photo-activated (4h) and imaged at 12 hours with either Lysotracker (upper panels) or DQ-BSA (lower panel) with enlargements (boxes) and Pearson’s coefficients (R(r)). Scale bars = 5 um.

Figure 3

mTOR reactivation inhibits autophagy and initiates lysosome reformation. (a) TEM (bottom, scale bar = 5 um) or fluorescence (top, scale bar, 10 um) of NRK-LC3 cells starved for hours (h) with enlargements (boxes). (b) and (d) immunoblots of (a) with indicated antibodies after transfection (d) with non-specific (NS) or atg5 RNAi. (c) Autophagy (black lines = independent experiments) and phospho-S6K (densitometry of b, red line). (e)-(g) Two hour starved LAMP1-YFP-NRK-LC3 cells treated with 100 nM rapamycin for another 4 (6 h) or 6 (8 h) hours analyzed by blotting (e) as in (b) or microscopy (f, scale bar, 5 um) with quantitation (g). Error bars show s.e.m (n=3).

Figure 4

Autophagic lysosome reformation. (a) LAMP1/LC3-NRK cells were starved for 4 hours and treated with GTPγS for 6 hours starvation. (b) Rab7Q67L-GFP transfected LC3-CFP/Lamp1-RFP-NRK cells starved for 10 hours. (c) /Rab7-CFP-NRK cells starved for 2 hrs and treated with 100 nM rapamycin for 8 hours starvation. (d) LAMP1-YFP/ NRK-LC3 cells starved for hours (h) with 1ug/ml leupeptin and blotted with indicated antibodies or (e) imaged. (f) Fibroblast cells from Scheie syndrome (GM01256) patients or controls starved and blotted with indicated antibodies or (g) imaged. Lysosome size classified by IPP software. (h) Provisional model of the autophagic lysosome reformation (ALR) cycle. Scale bars = 5 um.