Figure 5.
Figure 5. ATP13A2 regulation of APS–LYS fusion is HDAC6 dependent. (a and b) Reduced colocalization of endogenous LC3 and LAMP1 in ATP13A2-null HeLa cells. Control (WT) and ATP13A2-null (KO) HeLa cells were treated with DMSO (a, upper panels) or rapamycin for 16 h to induce autophagy (a, lower panels, Rapa). Immunodetection of endogenous LAMP1 (red) and LC3 (green) at lower (bar, 7.5 µm) and higher (insets; bar, 1.5 µm) magnification is shown (a). LC3-LAMP1 colocalization in the rapamycin-treated group is shown (b; WT/Rapa: n = 48, KO/Rapa: n = 59). ****, P < 0.0001. (c and d) Colocalization of GFP-mCherry-LC3 and LAMP1. Control (WT) and ATP13A2-null (KO) MEFs expressing GFP-mCherry-LC3 were immunodetected for LAMP1 (blue). Colocalization of GFP-mCherry-LC3 and LAMP1 is shown at low (merged image; bar, 7.5 µm) and high (zoom; bar, 1.5 µm) magnification (c). Representative histograms of colocalization are shown (d). Red–blue colocalization is seen in control MEFs; green–red colocalization is detected in ATP13A2-null MEFs (d). (e–l) In vitro reconstitution of APS–LYS fusion. APS and LYS were isolated from livers of starved ATP13A2-null (KO) mice or their control littermates (WT). APS (red) and LYS (green) were labeled with an anti-LC3B antibody and an anti-LAMP1 antibody, respectively. Fusion was assessed in energy-regenerating buffer supplied with various APS and LYS combinations. Fusion of both APS and LYS from WT mouse livers in the absence of ATP/GTP/creatine phosphokinase (e; No energy) or with (g; BafA1) or without (f) the fusion inhibitor bafilomycin A1 served as controls. Results of other indicated combinations are shown in h, i, and j. HDAC6 inhibitor tubacin was also included to show essential roles of HDAC6 in APS–LYS fusion (k). Bar, 7.5 µm; Zoom, bar, 1.5 µm. Quantitative analysis of fused versus total vesicles is shown (j). **, P < 0.01; ****, P < 0.0001, n = 4. (m and n) HDAC6 reverses LC3 accumulation in ATP13A2-null cells. Control (WT) and ATP13A2-null (KO) HEK293 cells expressing control plasmid (HDAC6-FLAG, −) or HDAC6-flag (HDAC6-FLAG, +) followed by treatment with DMSO (BafA1, –) or bafilomycin A1 (BafA1, +). LC3 (both LC3-I and LC3-II), HDAC6 (FLAG), and actin were immunodetected (m). Quantitative analysis is shown (n). **, P < 0.01, n = 3. (o and p) HDAC6 rescues ATP13A2-null impaired fusion. WT MEFs (WT) and ATP13A2-null MEFs (KO) cotransfected GFP-mCherry-LC3 reporter with either pcDNA3.1 (KO) or WT HDAC6 (HDAC6) were imaged for fusion. Both lower (bar, 7.5 µm) and higher (o; Zoom, bar, 1.5 µm) magnification images are shown. Analysis of nonfused (yellow) versus total vesicles is shown (p). ****, P < 0.0001. WT: n = 178, KO: n = 165, KO/HDAC6: n = 186.

ATP13A2 regulation of APS–LYS fusion is HDAC6 dependent. (a and b) Reduced colocalization of endogenous LC3 and LAMP1 in ATP13A2-null HeLa cells. Control (WT) and ATP13A2-null (KO) HeLa cells were treated with DMSO (a, upper panels) or rapamycin for 16 h to induce autophagy (a, lower panels, Rapa). Immunodetection of endogenous LAMP1 (red) and LC3 (green) at lower (bar, 7.5 µm) and higher (insets; bar, 1.5 µm) magnification is shown (a). LC3-LAMP1 colocalization in the rapamycin-treated group is shown (b; WT/Rapa: n = 48, KO/Rapa: n = 59). ****, P < 0.0001. (c and d) Colocalization of GFP-mCherry-LC3 and LAMP1. Control (WT) and ATP13A2-null (KO) MEFs expressing GFP-mCherry-LC3 were immunodetected for LAMP1 (blue). Colocalization of GFP-mCherry-LC3 and LAMP1 is shown at low (merged image; bar, 7.5 µm) and high (zoom; bar, 1.5 µm) magnification (c). Representative histograms of colocalization are shown (d). Red–blue colocalization is seen in control MEFs; green–red colocalization is detected in ATP13A2-null MEFs (d). (e–l) In vitro reconstitution of APS–LYS fusion. APS and LYS were isolated from livers of starved ATP13A2-null (KO) mice or their control littermates (WT). APS (red) and LYS (green) were labeled with an anti-LC3B antibody and an anti-LAMP1 antibody, respectively. Fusion was assessed in energy-regenerating buffer supplied with various APS and LYS combinations. Fusion of both APS and LYS from WT mouse livers in the absence of ATP/GTP/creatine phosphokinase (e; No energy) or with (g; BafA1) or without (f) the fusion inhibitor bafilomycin A1 served as controls. Results of other indicated combinations are shown in h, i, and j. HDAC6 inhibitor tubacin was also included to show essential roles of HDAC6 in APS–LYS fusion (k). Bar, 7.5 µm; Zoom, bar, 1.5 µm. Quantitative analysis of fused versus total vesicles is shown (j). **, P < 0.01; ****, P < 0.0001, n = 4. (m and n) HDAC6 reverses LC3 accumulation in ATP13A2-null cells. Control (WT) and ATP13A2-null (KO) HEK293 cells expressing control plasmid (HDAC6-FLAG, −) or HDAC6-flag (HDAC6-FLAG, +) followed by treatment with DMSO (BafA1, –) or bafilomycin A1 (BafA1, +). LC3 (both LC3-I and LC3-II), HDAC6 (FLAG), and actin were immunodetected (m). Quantitative analysis is shown (n). **, P < 0.01, n = 3. (o and p) HDAC6 rescues ATP13A2-null impaired fusion. WT MEFs (WT) and ATP13A2-null MEFs (KO) cotransfected GFP-mCherry-LC3 reporter with either pcDNA3.1 (KO) or WT HDAC6 (HDAC6) were imaged for fusion. Both lower (bar, 7.5 µm) and higher (o; Zoom, bar, 1.5 µm) magnification images are shown. Analysis of nonfused (yellow) versus total vesicles is shown (p). ****, P < 0.0001. WT: n = 178, KO: n = 165, KO/HDAC6: n = 186.

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