Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation - PubMed (original) (raw)

Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation

Yueguang Rong et al. Proc Natl Acad Sci U S A. 2011.

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Abstract

Autophagy is a conserved cellular process to degrade and recycle cytoplasmic components. During autophagy, lysosomes fuse with an autophagosome to form an autolysosome. Sequestered components are degraded by lysosomal hydrolases and presumably released into the cytosol by lysosomal efflux permeases. Following starvation-induced autophagy, lysosome homeostasis is restored by autophagic lysosome reformation (ALR) requiring activation of the "target of rapamycin" (TOR) kinase. Spinster (Spin) encodes a putative lysosomal efflux permease with the hallmarks of a sugar transporter. Drosophila spin mutants accumulate lysosomal carbohydrates and enlarged lysosomes. Here we show that defects in spin lead to the accumulation of enlarged autolysosomes. We find that spin is essential for mTOR reactivation and lysosome reformation following prolonged starvation. Further, we demonstrate that the sugar transporter activity of Spin is essential for ALR.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Spin colocalizes with the lysosomal membrane marker Lamp1, and defects in spin lead to enlargement of Lamp1-positive compartments. (A) NRK cells were transfected with Spin-GFP (green) and stained for Mitotracker (red, Left) or Lysotracker (red, Right). (Scale bar, 5 μm.) (B) NRK cells were transfected with Spin-GFP and Lamp1-Cherry. (Scale bar 5 μm.) (C) NRK cells were transfected with nonspecific (NS)- or _spin_-RNAi. After 2 d, cells were again transfected with NS- or _spin_-RNAi and Lamp1-YFP; 16 h after transfection, cells were starved for 0 or 10 h and observed by confocal microscopy. (Scale bar, 5 μm.) (D) NRK cells were transfected with nonspecific (NS)-RNAi or RNAi against spin. Two days after transfection, cells were retransfected with RNAi and either hSpin-CFP (Rescue)/Lamp1-YFP or Vector/Lamp1-YFP. Twenty-four hours after the second transfection, cells were starved for 10 h and then observed by confocal microscopy. (E) Cells from D were assessed in a blind fashion for rescue of the enlarged lysosome phenotype after starvation for 10 h and quantified. One hundred cells were counted. Error bars represent s.d. from more than three independent experiments. (F) Control Drosophila expressing _tub_-Lamp1-GFP versus transheterozygous _spin_10403/_spin_K09905 (spin mutant) larvae expressing _tub_-Lamp1-GFP were either fed or starved for 12 h, and lysosomes in fatbody cells were observed by fluorescent microscopy. Blue: Höescht. Green: Lamp1-GFP. (Scale bars, 20 μm.)

Fig. 2.

Fig. 2.

Enlargement of lysosomes upon spin knockdown is autophagy dependent. (A) NRK cells were transfected with CFP-LC3 (pseudocolored as green) and Spin-YFP (pseudocolored as red), and 24 h after transfection, cells were starved for 4 h. (Inset) A close-up of the discrete ring structure of Spin-YFP surrounding the LC3 assembly. (Scale bars, 5 μm.) (B) NRK cells were transfected with nonspecific (NS)- or _spin_-RNAi. After 2 d, cells were again transfected with NS- or _spin_-RNAi and Lamp1-YFP; 16 h after transfection, cells were starved for 10 h with or without the addition of 1 mM 3-MA, and observed by confocal microscopy. (Scale bars, 5 μm.) (C) NRK cells were transfected with nonspecific (NS)-RNAi, _spin_-RNAi, or _spin_-RNAi plus Beclin 1 (Bec)-RNAi. After 2 d, cells were again transfected with RNAi and Lamp1-YFP; 16 h after transfection, cells were starved for 0 or 10 h and observed by confocal microscopy. (Scale bars, 5 μm.) (D) Western blot for the Beclin-1 protein and Actin control for a representative sample of cells transfected with NS-RNAi plus _spin_-RNAi, or _spin_-RNAi plus _beclin 1_-RNAi. (E) Cells expressing nonspecific (NS)-RNAi, _spin_-RNAi, or _spin_-RNAi plus Beclin 1 (Bec)-RNAi were quantified in a blind fashion for the presence of enlarged lysosomes after 10 h of starvation. One hundred cells were counted. Error bars indicate the SD from more than three independent experiments.

Fig. 3.

Fig. 3.

spin is required for autophagic lysosome reformation. (A) NRK-CFP-LC3 (pseudocolored as green)-Lamp1-YFP (pseudocolored as red) stable cells were transfected with nonspecific (NS)- or _spin_-RNAi and starved for 0, 4, 8, or 12 h. (Scale bar, 5 μm.) (B) Cells from (A) were assessed in a blind fashion for change in number of Lamp1-positive structures relative to nonstarved cells. Fifty cells were counted per timepoint. Error bars represent S.D. derived from multiple experiments. (C) Nonspecific (NS)- or _spin_-RNAi-transfected NRK-CFP-LC3-Lamp1-YFP cells were starved for 8 h. Cells were then Z-stack scanned at 0.5-μm intervals, and images were collapsed to construct 3D models using IMRIS. Blue, LC3; Green, Lamp1; Red, Lysotracker. (Scale bars, 5 μm.) (D) NRK cells were transfected with nonspecific (NS) or _spin_-RNAi and after 2 d, cells were transfected with NS or _spin_-RNAi, CFP-Rab7, and Lamp1-YFP. Twenty-four hours after transfection, cells were starved for 0 or 12 h before imaging. (Scale bars, 5 μm.) (E) NS or _spin_-RNAi-transfected NRK cells were starved for 12 h, autolysosome/lysosome were purified, and the protein levels of Rab7 and Lamp2 were analyzed by Western blot. The signal intensity of Rab7 and Lamp2 were measured by IPP and the histogram shows the ratio between Rab7 and Lamp2.

Fig. 4.

Fig. 4.

Sugar transporter activity is essential for Spin's role in the regulation of autophagic lysosome reformation. (A) Carbohydrates accumulate in spin knockdown cells under prolonged starvation. NRK (CFP-LC3) cells were transfected with NS- or _spin_-RNAi. After 2 d, cells were starved for 18 h and subjected to periodic acid-Schiff (PAS) staining. (Scale bars, 5 μm.) Arrows indicate PAS-positive structures. Histogram shows the percentage of PAS-positive structures from multiple experiments counting a total of 100 cells, with mean and SD shown. (B) The alignment of the spin amino acid sequence from representative species shown in standard single letter code. The conserved glutamic acid residue (E) which is essential for sugar transporter activity is highlighted in red. (C) NRK cells were transfected with RNAi against spin. Two days after transfection, cells were retransfected with RNAi and vector/Lamp1-YFP, hSpin-CFP-wt/Lamp1-YFP or h_spin_-CFP-mutant (_spin_E217K)/Lamp1-YFP. Twenty-four hours after the second transfection, cells were starved for 10 h and then observed by confocal microscopy. (Scale bar, 5 μm.)

Fig. 5.

Fig. 5.

spin is required for mTOR reactivation following starvation. (A) NRK-LC3 cells were transfected with nonspecific RNAi (NS) or RNAi against spin. Sixty hours after transfection, cells were starved 0, 2, 6, or 8 h, harvested, and analyzed by Western blot using antibodies against Phospho-p70 S6 Kinase (P-S6K), Total S6 Kinase (S6K), and Actin. (B and C) NRK cells were transfected with _spin_-RNAi as in A, and starved for 10 h. FCS was then added back for 0–120 min; cells were harvested and analyzed by Western blot using antibodies against Phospho-p70 S6 Kinase (P-S6K) (B), or observed by confocal microscopy for the Lamp1 intracellular pattern (C). (Scale bar, 5 μm.) (Inset) Enlargement of tubules extruding from lysosomes.

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