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Soluble adenylyl cyclase is essential for proper lysosomal acidification

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ABSTRACT

Lysosomes are the main degradative compartment in cells and require an acidic luminal environment for correct function. Rahman et al. show that soluble adenylyl cyclase is required for localization of the V-ATPase proton pump to lysosomes and therefore lysosomal acidification and function.

No MeSH data available.


Related in: MedlinePlus

Electron-dense AVs accumulate in the absence of sAC activity. (A) AVs were subcategorized based on their morphology. AP: double membrane structures and/or double membrane structure containing undigested organelles; EAL: single membrane structures containing partially digested electron-dense material; LAL: single membrane structures containing amorphous electron-dense material. Double arrows represent double membrane, and single arrow represents single membrane. (B) Quantitative analysis of the number of AVs in WT, WT+KH7, and sAC KO MEFs. n = 10 cells/condition, two independent experiments. **, P < 0.02. (C) Representative electron micrographs of hippocampal dentate granule cells in WT and sAC KO aged mice. High-magnification views of the boxed areas below show examples of AVs in KO mice. (D) Quantitative analysis of the number of AVs in WT and sAC KO granular cells of the dentate gyrus. n = 3 mice/condition; n = 10 cells/mouse. ***, P < 0.002. (E) Representative electron micrographs of the pyramidal cells of the CA1 in WT and sAC KO aged mice. High-magnification views of the boxed areas below show examples of AVs in KO mice. Bars: (A, C [bottom], and E [bottom]) 500 nm; (C and E, top) 2 µm. (F) Quantitative analysis of the number of vacuoles in WT and sAC KO pyramidal cells of the CA1 of the hippocampus. n = 3 mice/condition; n = 10 cells/mouse. *, P < 0.05. All values are given as mean ± SEM.
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fig6: Electron-dense AVs accumulate in the absence of sAC activity. (A) AVs were subcategorized based on their morphology. AP: double membrane structures and/or double membrane structure containing undigested organelles; EAL: single membrane structures containing partially digested electron-dense material; LAL: single membrane structures containing amorphous electron-dense material. Double arrows represent double membrane, and single arrow represents single membrane. (B) Quantitative analysis of the number of AVs in WT, WT+KH7, and sAC KO MEFs. n = 10 cells/condition, two independent experiments. **, P < 0.02. (C) Representative electron micrographs of hippocampal dentate granule cells in WT and sAC KO aged mice. High-magnification views of the boxed areas below show examples of AVs in KO mice. (D) Quantitative analysis of the number of AVs in WT and sAC KO granular cells of the dentate gyrus. n = 3 mice/condition; n = 10 cells/mouse. ***, P < 0.002. (E) Representative electron micrographs of the pyramidal cells of the CA1 in WT and sAC KO aged mice. High-magnification views of the boxed areas below show examples of AVs in KO mice. Bars: (A, C [bottom], and E [bottom]) 500 nm; (C and E, top) 2 µm. (F) Quantitative analysis of the number of vacuoles in WT and sAC KO pyramidal cells of the CA1 of the hippocampus. n = 3 mice/condition; n = 10 cells/mouse. *, P < 0.05. All values are given as mean ± SEM.

Mentions: If the AV clearance defect is a direct consequence of diminished proteolytic activity in sAC KO cells, the absence of sAC should result in increased accumulation of APs and early-stage ALs (EALs; i.e., single membrane structures containing partially digested electron dense material) but not late-stage ALs (LALs; i.e., single membrane structures containing amorphous electron dense material; Fig. 6 A; Lee et al., 2010). Consistent with our hypothesis, ultrastructural analysis of sAC KO and WT MEFs treated with KH7 revealed an increase in both APs and EALs relative to untreated WT MEFs (Fig. 6 B). In WT MEFs treated with KH7, where sAC activity is acutely inhibited, this increase in APs and EALs was accompanied by a decrease in LALs, whereas in sAC KO MEFs LAL accumulation was unchanged relative to WT MEFs (Fig. 6 B). Therefore, our data suggest that the sAC KO lysosomal pH and proteolysis defects lead to impaired clearance of AVs.


Soluble adenylyl cyclase is essential for proper lysosomal acidification
Electron-dense AVs accumulate in the absence of sAC activity. (A) AVs were subcategorized based on their morphology. AP: double membrane structures and/or double membrane structure containing undigested organelles; EAL: single membrane structures containing partially digested electron-dense material; LAL: single membrane structures containing amorphous electron-dense material. Double arrows represent double membrane, and single arrow represents single membrane. (B) Quantitative analysis of the number of AVs in WT, WT+KH7, and sAC KO MEFs. n = 10 cells/condition, two independent experiments. **, P < 0.02. (C) Representative electron micrographs of hippocampal dentate granule cells in WT and sAC KO aged mice. High-magnification views of the boxed areas below show examples of AVs in KO mice. (D) Quantitative analysis of the number of AVs in WT and sAC KO granular cells of the dentate gyrus. n = 3 mice/condition; n = 10 cells/mouse. ***, P < 0.002. (E) Representative electron micrographs of the pyramidal cells of the CA1 in WT and sAC KO aged mice. High-magnification views of the boxed areas below show examples of AVs in KO mice. Bars: (A, C [bottom], and E [bottom]) 500 nm; (C and E, top) 2 µm. (F) Quantitative analysis of the number of vacuoles in WT and sAC KO pyramidal cells of the CA1 of the hippocampus. n = 3 mice/condition; n = 10 cells/mouse. *, P < 0.05. All values are given as mean ± SEM.
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fig6: Electron-dense AVs accumulate in the absence of sAC activity. (A) AVs were subcategorized based on their morphology. AP: double membrane structures and/or double membrane structure containing undigested organelles; EAL: single membrane structures containing partially digested electron-dense material; LAL: single membrane structures containing amorphous electron-dense material. Double arrows represent double membrane, and single arrow represents single membrane. (B) Quantitative analysis of the number of AVs in WT, WT+KH7, and sAC KO MEFs. n = 10 cells/condition, two independent experiments. **, P < 0.02. (C) Representative electron micrographs of hippocampal dentate granule cells in WT and sAC KO aged mice. High-magnification views of the boxed areas below show examples of AVs in KO mice. (D) Quantitative analysis of the number of AVs in WT and sAC KO granular cells of the dentate gyrus. n = 3 mice/condition; n = 10 cells/mouse. ***, P < 0.002. (E) Representative electron micrographs of the pyramidal cells of the CA1 in WT and sAC KO aged mice. High-magnification views of the boxed areas below show examples of AVs in KO mice. Bars: (A, C [bottom], and E [bottom]) 500 nm; (C and E, top) 2 µm. (F) Quantitative analysis of the number of vacuoles in WT and sAC KO pyramidal cells of the CA1 of the hippocampus. n = 3 mice/condition; n = 10 cells/mouse. *, P < 0.05. All values are given as mean ± SEM.
Mentions: If the AV clearance defect is a direct consequence of diminished proteolytic activity in sAC KO cells, the absence of sAC should result in increased accumulation of APs and early-stage ALs (EALs; i.e., single membrane structures containing partially digested electron dense material) but not late-stage ALs (LALs; i.e., single membrane structures containing amorphous electron dense material; Fig. 6 A; Lee et al., 2010). Consistent with our hypothesis, ultrastructural analysis of sAC KO and WT MEFs treated with KH7 revealed an increase in both APs and EALs relative to untreated WT MEFs (Fig. 6 B). In WT MEFs treated with KH7, where sAC activity is acutely inhibited, this increase in APs and EALs was accompanied by a decrease in LALs, whereas in sAC KO MEFs LAL accumulation was unchanged relative to WT MEFs (Fig. 6 B). Therefore, our data suggest that the sAC KO lysosomal pH and proteolysis defects lead to impaired clearance of AVs.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Lysosomes are the main degradative compartment in cells and require an acidic luminal environment for correct function. Rahman et al. show that soluble adenylyl cyclase is required for localization of the V-ATPase proton pump to lysosomes and therefore lysosomal acidification and function.

No MeSH data available.


Related in: MedlinePlus