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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

sAC is essential for organellar acidification in primary neurons and human Huh7 cells. (A) Representative images of staining with LysoTracker (red) in WT or sAC KO 12 DIV neurons grown in the absence or presence of 30 µM KH7 (3 h), with 100 nM LysoTracker included for the final 30 min. (B) Quantified LysoTracker intensity in neurons. (C) Representative images of staining with LysoTracker (red) in human Huh7 cells treated with 30 µM KH7 (or 30 µM KH7.15) for 10 h, with 100 nM LysoTracker included for the final 30 min. (D) Quantified LysoTracker intensity in Huh7 cells. For B and D, LysoTracker intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.002.
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fig3: sAC is essential for organellar acidification in primary neurons and human Huh7 cells. (A) Representative images of staining with LysoTracker (red) in WT or sAC KO 12 DIV neurons grown in the absence or presence of 30 µM KH7 (3 h), with 100 nM LysoTracker included for the final 30 min. (B) Quantified LysoTracker intensity in neurons. (C) Representative images of staining with LysoTracker (red) in human Huh7 cells treated with 30 µM KH7 (or 30 µM KH7.15) for 10 h, with 100 nM LysoTracker included for the final 30 min. (D) Quantified LysoTracker intensity in Huh7 cells. For B and D, LysoTracker intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.002.

Mentions: Lysosomal dysfunction and defective autophagy have been linked to age-related neurodegenerative diseases, which arise when neurons erroneously accumulate long-lived or misfolded proteins and damaged organelles (Menzies et al., 2015). Therefore, we asked whether sAC is essential for lysosomal acidification in neurons. To examine organelle acidification in primary neurons, which proved to be unsuitable for use with dextran conjugates, we took advantage of LysoTracker, a qualitative acidic pH indicator dye, which is enriched and strongly fluoresces in compartments that have a pH below 5 (Lee et al., 2010). We first confirmed that LysoTracker reproduced the lysosomal acidification defect observed in sAC KO MEFs. As a qualitative marker of organelle acidity, we limited our use of LysoTracker to compare overall fluorescence intensity or number of acidic puncta within a cell line before and after pharmacological treatment. In WT MEFs, the number of LysoTracker puncta (Fig. S4) and overall intensity (not depicted) was unaffected by cAMP addition. In contrast, cAMP increased the number of LysoTracker-positive puncta (Fig. S4) and overall intensity (not depicted) in sAC KO MEFs. Thus, LysoTracker staining reflects the ability of cAMP to rescue the lysosomal acidification defect in sAC KO cells demonstrated quantitatively above (Fig. 1, C and E). LysoTracker intensity of 12 DIV WT primary neurons was diminished when sAC was inhibited using KH7 (Fig. 3, A and B). These effects of KH7 were specifically mediated via inhibition of sAC activity because the same treatment was inert on 12 DIV sAC KO neurons. KH7, but not the inert structurally related KH7.15 (Wu et al., 2006), also diminished LysoTracker intensity in a human liver cancer cell line, Huh7 (Fig. 3, C and D). These data demonstrate that sAC regulates organellar acidification in different cell types and in mouse and human cells and is most likely a general phenomenon. These data also suggest that sAC’s role in organellar acidification may expand beyond acidifying endosomes into lysosomes. The quantitative dextran conjugate method is biased toward those lysosomes actively involved in the endocytotic pathway, whereas LysoTracker identifies all acidic vesicles.


Soluble adenylyl cyclase is essential for proper lysosomal acidification
sAC is essential for organellar acidification in primary neurons and human Huh7 cells. (A) Representative images of staining with LysoTracker (red) in WT or sAC KO 12 DIV neurons grown in the absence or presence of 30 µM KH7 (3 h), with 100 nM LysoTracker included for the final 30 min. (B) Quantified LysoTracker intensity in neurons. (C) Representative images of staining with LysoTracker (red) in human Huh7 cells treated with 30 µM KH7 (or 30 µM KH7.15) for 10 h, with 100 nM LysoTracker included for the final 30 min. (D) Quantified LysoTracker intensity in Huh7 cells. For B and D, LysoTracker intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.002.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig3: sAC is essential for organellar acidification in primary neurons and human Huh7 cells. (A) Representative images of staining with LysoTracker (red) in WT or sAC KO 12 DIV neurons grown in the absence or presence of 30 µM KH7 (3 h), with 100 nM LysoTracker included for the final 30 min. (B) Quantified LysoTracker intensity in neurons. (C) Representative images of staining with LysoTracker (red) in human Huh7 cells treated with 30 µM KH7 (or 30 µM KH7.15) for 10 h, with 100 nM LysoTracker included for the final 30 min. (D) Quantified LysoTracker intensity in Huh7 cells. For B and D, LysoTracker intensity was quantified using MetaMorph in multiple cells from three independent experiments. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.002.
Mentions: Lysosomal dysfunction and defective autophagy have been linked to age-related neurodegenerative diseases, which arise when neurons erroneously accumulate long-lived or misfolded proteins and damaged organelles (Menzies et al., 2015). Therefore, we asked whether sAC is essential for lysosomal acidification in neurons. To examine organelle acidification in primary neurons, which proved to be unsuitable for use with dextran conjugates, we took advantage of LysoTracker, a qualitative acidic pH indicator dye, which is enriched and strongly fluoresces in compartments that have a pH below 5 (Lee et al., 2010). We first confirmed that LysoTracker reproduced the lysosomal acidification defect observed in sAC KO MEFs. As a qualitative marker of organelle acidity, we limited our use of LysoTracker to compare overall fluorescence intensity or number of acidic puncta within a cell line before and after pharmacological treatment. In WT MEFs, the number of LysoTracker puncta (Fig. S4) and overall intensity (not depicted) was unaffected by cAMP addition. In contrast, cAMP increased the number of LysoTracker-positive puncta (Fig. S4) and overall intensity (not depicted) in sAC KO MEFs. Thus, LysoTracker staining reflects the ability of cAMP to rescue the lysosomal acidification defect in sAC KO cells demonstrated quantitatively above (Fig. 1, C and E). LysoTracker intensity of 12 DIV WT primary neurons was diminished when sAC was inhibited using KH7 (Fig. 3, A and B). These effects of KH7 were specifically mediated via inhibition of sAC activity because the same treatment was inert on 12 DIV sAC KO neurons. KH7, but not the inert structurally related KH7.15 (Wu et al., 2006), also diminished LysoTracker intensity in a human liver cancer cell line, Huh7 (Fig. 3, C and D). These data demonstrate that sAC regulates organellar acidification in different cell types and in mouse and human cells and is most likely a general phenomenon. These data also suggest that sAC’s role in organellar acidification may expand beyond acidifying endosomes into lysosomes. The quantitative dextran conjugate method is biased toward those lysosomes actively involved in the endocytotic pathway, whereas LysoTracker identifies all acidic vesicles.

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