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Results: 4

1.
Figure 1

Figure 1. From: Autophagy termination and lysosome reformation regulated by mTOR.

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.

Li Yu, et al. Nature. ;465(7300):942-946.
2.
Figure 4

Figure 4. From: Autophagy termination and lysosome reformation regulated by mTOR.

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.

Li Yu, et al. Nature. ;465(7300):942-946.
3.
Figure 2

Figure 2. From: Autophagy termination and lysosome reformation regulated by mTOR.

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.

Li Yu, et al. Nature. ;465(7300):942-946.
4.
Figure 3

Figure 3. From: Autophagy termination and lysosome reformation regulated by mTOR.

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).

Li Yu, et al. Nature. ;465(7300):942-946.

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