Figure 4. HDAC6-dependent LYS accumulation with ATP13A2 deficiency. (a and b) HDAC6 reverses LYS accumulation in ATP13A2-null MEFs. LYS in ATP13A2-null (KO/Ctrl) and control MEF cells (WT) were immunodetected for LAMP1 (green). Expression of HDAC6 (KO/HDAC6 WT), but not H664A mutant (KO/HDAC6 H664A), inhibited LYS accumulation in ATP13A2-null cells. Quantification of LAMP1-positive structures by ImageJ is shown (b). Bar, 10 µm. **, P < 0.01. WT: n = 36, KO: n = 41, KO/HDAC6 WT: n = 39, KO/HDAC6 H664A: n = 46. (c and d) ATP13A2-regulated α-tubulin acetylation is HDAC6 dependent. α-Tubulin acetylation (Ac-tub, green) was detected in WT MEFs (WT), ATP13A2-null MEFs (KO/Ctrl), ATP13A2-null MEFs expressing HDAC6 (KO/HDAC6 WT), and ATP13A2-null MEFs expressing HDAC6 H611A (KO/HDAC6 H611A). HDAC6 variants were detected with an anti-FLAG antibody (red). Cell boundary was labeled by dashed lines. Bar, 5 µm (c and e). Quantification of Ac-tub intensity per cell was done with ImageJ. **, P < 0.01; ***, P < 0.001. WT: n = 41, KO: n = 38, KO/HDAC6 WT: n = 31, KO/HDAC6 H611A: n = 46. (e) Alignment of human and Drosophila HDAC6 (Fly) catalytic domain. Red indicates the active histidine (H). (f and g) dHDAC6 reduces tubulin acetylation in dATP13A2 RNAi fly. Fly brain lysates were immunodetected for acetylated tubulin (Ac-tub) and total tubulin (α-tub). WT fly expressing elav-gal4 alone (Elav/+) and dATP13A2 RNAi fly (RNAi) expressing elav-gal4 alone (Ctrl), dHDAC6 (dHDAC6 WT), or dHDAC6 H664A mutant (dHDAC6 H664A) were analyzed (d). Quantitative analysis is shown (e). ***, P < 0.001, n = 3. (h–j) dATP13A2 RNAi caused dHDAC6 dependent LYS accumulation. Fly follicle cells were probed with LysoTracker (upper panel) and active cathepsin B (Active Cath B, lower panel). Control Da-gal4 expressor (Da/+) and dATP13A2 RNAi line (RNAi) expressing Da-gal4 alone (Ctrl), dHDAC6 (dHDAC6 WT), or dHDAC6 H664A mutant (dHDAC6 H664A) (f). Quantitative analysis of LysoTracker intensity (g) and active cathepsin B (h) are shown. Bar, 10 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For Lysotracker: Da/+: n = 7, Ctrl: n = 6, dHDAC6 WT: n = 7, dHDAC6 H664A: n = 9. For active CathB: Da/+: n = 7, Ctrl: n = 6, dHDAC6 WT: n = 7, dHDAC6 H664A: n = 6. (k and l) dATP13A2 RNAi causes dHDAC6-dependent LAMP1 increase. Fly head lysates were immunodetected for LAMP1 and actin. The control elav-gal4 expressor (Elav/+) and dATP13A2 RNAi line (RNAi) expressing either elav-gal4 alone (Ctrl), WT dHDAC6 (dHDAC6 WT), or dHDAC6 H664A (dHDAC6 H664A) were analyzed (i). Quantitative analysis is shown (j; n = 3). ***, P < 0.001; ****, P < 0.0001. (m and n) TEM analysis of LYS. WT, ATP13A2-null HEK293 cells (KO/Ctrl) and their HDAC6 expressors (KO/HDAC6) were starved and analyzed by TEM. Normal LYS (yellow arrowheads) and abnormal LYS (white arrowheads) are indicated. Bar, 1 µm. Quantitation of large LYS (diameter, >0.45 µm) is shown (n). ***, P < 0.001. WT: n = 21, KO/Ctrl: n = 32, KO/HDAC6: n = 26.
Figure 4.

HDAC6-dependent LYS accumulation with ATP13A2 deficiency. (a and b) HDAC6 reverses LYS accumulation in ATP13A2-null MEFs. LYS in ATP13A2-null (KO/Ctrl) and control MEF cells (WT) were immunodetected for LAMP1 (green). Expression of HDAC6 (KO/HDAC6 WT), but not H664A mutant (KO/HDAC6 H664A), inhibited LYS accumulation in ATP13A2-null cells. Quantification of LAMP1-positive structures by ImageJ is shown (b). Bar, 10 µm. **, P < 0.01. WT: n = 36, KO: n = 41, KO/HDAC6 WT: n = 39, KO/HDAC6 H664A: n = 46. (c and d) ATP13A2-regulated α-tubulin acetylation is HDAC6 dependent. α-Tubulin acetylation (Ac-tub, green) was detected in WT MEFs (WT), ATP13A2-null MEFs (KO/Ctrl), ATP13A2-null MEFs expressing HDAC6 (KO/HDAC6 WT), and ATP13A2-null MEFs expressing HDAC6 H611A (KO/HDAC6 H611A). HDAC6 variants were detected with an anti-FLAG antibody (red). Cell boundary was labeled by dashed lines. Bar, 5 µm (c and e). Quantification of Ac-tub intensity per cell was done with ImageJ. **, P < 0.01; ***, P < 0.001. WT: n = 41, KO: n = 38, KO/HDAC6 WT: n = 31, KO/HDAC6 H611A: n = 46. (e) Alignment of human and Drosophila HDAC6 (Fly) catalytic domain. Red indicates the active histidine (H). (f and g) dHDAC6 reduces tubulin acetylation in dATP13A2 RNAi fly. Fly brain lysates were immunodetected for acetylated tubulin (Ac-tub) and total tubulin (α-tub). WT fly expressing elav-gal4 alone (Elav/+) and dATP13A2 RNAi fly (RNAi) expressing elav-gal4 alone (Ctrl), dHDAC6 (dHDAC6 WT), or dHDAC6 H664A mutant (dHDAC6 H664A) were analyzed (d). Quantitative analysis is shown (e). ***, P < 0.001, n = 3. (h–j) dATP13A2 RNAi caused dHDAC6 dependent LYS accumulation. Fly follicle cells were probed with LysoTracker (upper panel) and active cathepsin B (Active Cath B, lower panel). Control Da-gal4 expressor (Da/+) and dATP13A2 RNAi line (RNAi) expressing Da-gal4 alone (Ctrl), dHDAC6 (dHDAC6 WT), or dHDAC6 H664A mutant (dHDAC6 H664A) (f). Quantitative analysis of LysoTracker intensity (g) and active cathepsin B (h) are shown. Bar, 10 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For Lysotracker: Da/+: n = 7, Ctrl: n = 6, dHDAC6 WT: n = 7, dHDAC6 H664A: n = 9. For active CathB: Da/+: n = 7, Ctrl: n = 6, dHDAC6 WT: n = 7, dHDAC6 H664A: n = 6. (k and l) dATP13A2 RNAi causes dHDAC6-dependent LAMP1 increase. Fly head lysates were immunodetected for LAMP1 and actin. The control elav-gal4 expressor (Elav/+) and dATP13A2 RNAi line (RNAi) expressing either elav-gal4 alone (Ctrl), WT dHDAC6 (dHDAC6 WT), or dHDAC6 H664A (dHDAC6 H664A) were analyzed (i). Quantitative analysis is shown (j; n = 3). ***, P < 0.001; ****, P < 0.0001. (m and n) TEM analysis of LYS. WT, ATP13A2-null HEK293 cells (KO/Ctrl) and their HDAC6 expressors (KO/HDAC6) were starved and analyzed by TEM. Normal LYS (yellow arrowheads) and abnormal LYS (white arrowheads) are indicated. Bar, 1 µm. Quantitation of large LYS (diameter, >0.45 µm) is shown (n). ***, P < 0.001. WT: n = 21, KO/Ctrl: n = 32, KO/HDAC6: n = 26.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal