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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.


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In the absence of sAC activity, lysosomal pH is elevated. (A) Representative images of WT or sAC KO 3T3 MEFs loaded with dextran beads conjugated to pH-insensitive rhodamine (red) and pH-sensitive fluorescein (green). Bar, 10 µm. (B) Frequency distribution of lysosomal pH measured as fluorescein/rhodamine ratios of individual lysosomes in WT and sAC KO 3T3 MEFs. Lysosomal pH values were determined from calibration curves generated from permeabilized cells in various pH buffers (Fig. S2). Number of lysosomes counted: WT = 3,141, KO = 3,559, n = 18 (from six independent experiment days). (C) Mean lysosomal pH in WT and sAC KO 3T3 MEFs in the absence or presence of KH7 or cAMP calculated from data shown in B, D, and E. (D) Frequency distribution of lysosomal pH in WT (WT+KH7) and sAC KO (KO+KH7) 3T3 MEFs treated with 30 µM KH7 for 10 h. Shown for comparison are the frequency distribution curves of WT and sAC KO MEFs from B. Number of lysosomes counted: WT+KH7 = 2,036, n = 12; KO+KH7 = 395, n = 3. (E) Frequency distribution of lysosomal pH in WT (WT+cAMP) and sAC KO (KO+cAMP) 3T3 MEFs treated with 500 µM Sp-8-cpt-cAMP for 60 min. Shown for comparison are the frequency distribution curves of WT and sAC KO MEFs from B. Number of lysosomes counted: KO+cAMP = 2,003, n = 12; WT+cAMP = 579, n = 3. (F) Frequency distribution of lysosomal pH in WT and sAC KO SV40 MEFs. Number of lysosomes counted: WT = 904, sAC KO =1,089, n = 6. (G) Mean lysosomal pH in WT and sAC KO SV40 MEFs calculated from data shown in F. (H) Frequency distribution of lysosomal pH in sAC KO 3T3 MEFs alone (KO), treated with 500 µM Sp-8-cpt-cAMP (KO+cAMP), or treated with 500 µM Sp-8-cpt-cAMP in the presence of 50 µM PKA inhibitor, KT 5720 (KO+cAMP+KT). Number of lysosomes counted: KO = 442, KO+cAMP = 250, KO+cAMP+KT = 1,024, n = 3 (from two independent experiment days). Data are distinct from the experiments shown in A–E. (I) Mean lysosomal pH in sAC KO 3T3 MEFs calculated from data shown in H. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig1: In the absence of sAC activity, lysosomal pH is elevated. (A) Representative images of WT or sAC KO 3T3 MEFs loaded with dextran beads conjugated to pH-insensitive rhodamine (red) and pH-sensitive fluorescein (green). Bar, 10 µm. (B) Frequency distribution of lysosomal pH measured as fluorescein/rhodamine ratios of individual lysosomes in WT and sAC KO 3T3 MEFs. Lysosomal pH values were determined from calibration curves generated from permeabilized cells in various pH buffers (Fig. S2). Number of lysosomes counted: WT = 3,141, KO = 3,559, n = 18 (from six independent experiment days). (C) Mean lysosomal pH in WT and sAC KO 3T3 MEFs in the absence or presence of KH7 or cAMP calculated from data shown in B, D, and E. (D) Frequency distribution of lysosomal pH in WT (WT+KH7) and sAC KO (KO+KH7) 3T3 MEFs treated with 30 µM KH7 for 10 h. Shown for comparison are the frequency distribution curves of WT and sAC KO MEFs from B. Number of lysosomes counted: WT+KH7 = 2,036, n = 12; KO+KH7 = 395, n = 3. (E) Frequency distribution of lysosomal pH in WT (WT+cAMP) and sAC KO (KO+cAMP) 3T3 MEFs treated with 500 µM Sp-8-cpt-cAMP for 60 min. Shown for comparison are the frequency distribution curves of WT and sAC KO MEFs from B. Number of lysosomes counted: KO+cAMP = 2,003, n = 12; WT+cAMP = 579, n = 3. (F) Frequency distribution of lysosomal pH in WT and sAC KO SV40 MEFs. Number of lysosomes counted: WT = 904, sAC KO =1,089, n = 6. (G) Mean lysosomal pH in WT and sAC KO SV40 MEFs calculated from data shown in F. (H) Frequency distribution of lysosomal pH in sAC KO 3T3 MEFs alone (KO), treated with 500 µM Sp-8-cpt-cAMP (KO+cAMP), or treated with 500 µM Sp-8-cpt-cAMP in the presence of 50 µM PKA inhibitor, KT 5720 (KO+cAMP+KT). Number of lysosomes counted: KO = 442, KO+cAMP = 250, KO+cAMP+KT = 1,024, n = 3 (from two independent experiment days). Data are distinct from the experiments shown in A–E. (I) Mean lysosomal pH in sAC KO 3T3 MEFs calculated from data shown in H. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: We derived MEF cell lines from sAC KO mice and from their WT littermates (Ramos-Espiritu et al., 2016). sAC KO 3T3 MEFs (hereafter referred to as sAC KO MEFs) generate less cAMP than WT MEFs, and unlike in WT MEFs, all the cAMP generated in sAC KO MEFs is insensitive to the sAC-specific inhibitor KH7 (Fig. S1). To quantify the pH of individual lysosomes in these MEF lines, we used ratiometric imaging of fluorescein (pH sensitive) and rhodamine (pH insensitive) dextran beads (Majumdar et al., 2007). The pH-sensitive nature of this dual-emission probe is demonstrated in Fig. S2. With decreasing pH, the pH-sensitive fluorescein (green) fluorescence is quenched, while the pH-insensitive rhodamine (red) fluorescence remains unchanged (Fig. S2, A and B); thus, the green/red ratio identifies each lysosome and determines its specific pH (Fig. S2 C). We confirmed the ability of this ratiometric method to measure changes in lysosomal pH by demonstrating alkalinization of lysosomes in WT cells perfused with 20 mM NH4Cl (Fig. S2, D and E). Using this quantitative method, lysosomal pH in WT MEFs was found to average 4.7 ± 0.1, consistent with previously published studies (Fig. 1, A–C; Ohkuma and Poole, 1978; Lee et al., 2010; Cang et al., 2013). In contrast, the mean pH of lysosomes in sAC KO MEFs was significantly higher, with their mean pH of 5.3 ± 0.1 (P < 0.001). In addition to having a higher mean pH, the distribution of pH values across all lysosomes was broader in sAC KO cells (Fig. 1 B).


Soluble adenylyl cyclase is essential for proper lysosomal acidification
In the absence of sAC activity, lysosomal pH is elevated. (A) Representative images of WT or sAC KO 3T3 MEFs loaded with dextran beads conjugated to pH-insensitive rhodamine (red) and pH-sensitive fluorescein (green). Bar, 10 µm. (B) Frequency distribution of lysosomal pH measured as fluorescein/rhodamine ratios of individual lysosomes in WT and sAC KO 3T3 MEFs. Lysosomal pH values were determined from calibration curves generated from permeabilized cells in various pH buffers (Fig. S2). Number of lysosomes counted: WT = 3,141, KO = 3,559, n = 18 (from six independent experiment days). (C) Mean lysosomal pH in WT and sAC KO 3T3 MEFs in the absence or presence of KH7 or cAMP calculated from data shown in B, D, and E. (D) Frequency distribution of lysosomal pH in WT (WT+KH7) and sAC KO (KO+KH7) 3T3 MEFs treated with 30 µM KH7 for 10 h. Shown for comparison are the frequency distribution curves of WT and sAC KO MEFs from B. Number of lysosomes counted: WT+KH7 = 2,036, n = 12; KO+KH7 = 395, n = 3. (E) Frequency distribution of lysosomal pH in WT (WT+cAMP) and sAC KO (KO+cAMP) 3T3 MEFs treated with 500 µM Sp-8-cpt-cAMP for 60 min. Shown for comparison are the frequency distribution curves of WT and sAC KO MEFs from B. Number of lysosomes counted: KO+cAMP = 2,003, n = 12; WT+cAMP = 579, n = 3. (F) Frequency distribution of lysosomal pH in WT and sAC KO SV40 MEFs. Number of lysosomes counted: WT = 904, sAC KO =1,089, n = 6. (G) Mean lysosomal pH in WT and sAC KO SV40 MEFs calculated from data shown in F. (H) Frequency distribution of lysosomal pH in sAC KO 3T3 MEFs alone (KO), treated with 500 µM Sp-8-cpt-cAMP (KO+cAMP), or treated with 500 µM Sp-8-cpt-cAMP in the presence of 50 µM PKA inhibitor, KT 5720 (KO+cAMP+KT). Number of lysosomes counted: KO = 442, KO+cAMP = 250, KO+cAMP+KT = 1,024, n = 3 (from two independent experiment days). Data are distinct from the experiments shown in A–E. (I) Mean lysosomal pH in sAC KO 3T3 MEFs calculated from data shown in H. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig1: In the absence of sAC activity, lysosomal pH is elevated. (A) Representative images of WT or sAC KO 3T3 MEFs loaded with dextran beads conjugated to pH-insensitive rhodamine (red) and pH-sensitive fluorescein (green). Bar, 10 µm. (B) Frequency distribution of lysosomal pH measured as fluorescein/rhodamine ratios of individual lysosomes in WT and sAC KO 3T3 MEFs. Lysosomal pH values were determined from calibration curves generated from permeabilized cells in various pH buffers (Fig. S2). Number of lysosomes counted: WT = 3,141, KO = 3,559, n = 18 (from six independent experiment days). (C) Mean lysosomal pH in WT and sAC KO 3T3 MEFs in the absence or presence of KH7 or cAMP calculated from data shown in B, D, and E. (D) Frequency distribution of lysosomal pH in WT (WT+KH7) and sAC KO (KO+KH7) 3T3 MEFs treated with 30 µM KH7 for 10 h. Shown for comparison are the frequency distribution curves of WT and sAC KO MEFs from B. Number of lysosomes counted: WT+KH7 = 2,036, n = 12; KO+KH7 = 395, n = 3. (E) Frequency distribution of lysosomal pH in WT (WT+cAMP) and sAC KO (KO+cAMP) 3T3 MEFs treated with 500 µM Sp-8-cpt-cAMP for 60 min. Shown for comparison are the frequency distribution curves of WT and sAC KO MEFs from B. Number of lysosomes counted: KO+cAMP = 2,003, n = 12; WT+cAMP = 579, n = 3. (F) Frequency distribution of lysosomal pH in WT and sAC KO SV40 MEFs. Number of lysosomes counted: WT = 904, sAC KO =1,089, n = 6. (G) Mean lysosomal pH in WT and sAC KO SV40 MEFs calculated from data shown in F. (H) Frequency distribution of lysosomal pH in sAC KO 3T3 MEFs alone (KO), treated with 500 µM Sp-8-cpt-cAMP (KO+cAMP), or treated with 500 µM Sp-8-cpt-cAMP in the presence of 50 µM PKA inhibitor, KT 5720 (KO+cAMP+KT). Number of lysosomes counted: KO = 442, KO+cAMP = 250, KO+cAMP+KT = 1,024, n = 3 (from two independent experiment days). Data are distinct from the experiments shown in A–E. (I) Mean lysosomal pH in sAC KO 3T3 MEFs calculated from data shown in H. All values are given as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: We derived MEF cell lines from sAC KO mice and from their WT littermates (Ramos-Espiritu et al., 2016). sAC KO 3T3 MEFs (hereafter referred to as sAC KO MEFs) generate less cAMP than WT MEFs, and unlike in WT MEFs, all the cAMP generated in sAC KO MEFs is insensitive to the sAC-specific inhibitor KH7 (Fig. S1). To quantify the pH of individual lysosomes in these MEF lines, we used ratiometric imaging of fluorescein (pH sensitive) and rhodamine (pH insensitive) dextran beads (Majumdar et al., 2007). The pH-sensitive nature of this dual-emission probe is demonstrated in Fig. S2. With decreasing pH, the pH-sensitive fluorescein (green) fluorescence is quenched, while the pH-insensitive rhodamine (red) fluorescence remains unchanged (Fig. S2, A and B); thus, the green/red ratio identifies each lysosome and determines its specific pH (Fig. S2 C). We confirmed the ability of this ratiometric method to measure changes in lysosomal pH by demonstrating alkalinization of lysosomes in WT cells perfused with 20 mM NH4Cl (Fig. S2, D and E). Using this quantitative method, lysosomal pH in WT MEFs was found to average 4.7 ± 0.1, consistent with previously published studies (Fig. 1, A–C; Ohkuma and Poole, 1978; Lee et al., 2010; Cang et al., 2013). In contrast, the mean pH of lysosomes in sAC KO MEFs was significantly higher, with their mean pH of 5.3 ± 0.1 (P < 0.001). In addition to having a higher mean pH, the distribution of pH values across all lysosomes was broader in sAC KO cells (Fig. 1 B).

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