Journal: Human Molecular Genetics

Article Title: Hexokinase activity is required for recruitment of parkin to depolarized mitochondria

doi: 10.1093/hmg/ddt407

Figure Lengend Snippet: Kinase active HK2 promotes depolarization-induced parkin recruitment in HeLa cells and primary cortical neurons. (A) HeLa lines stably expressing YFP-parkin, MitoDsRed2 and either control (left panels) or HK2 (right panels) shRNA transfected with V5-tagged LacZ (upper panels), HK2 WT (middle panels) or HK2 KD (lower panels) constructs and treated with 2 μm CCCP (2 h, low-glucose media). Scale bar: 20 μm. (B) Quantitation from (A) of the mean overlap area between YFP-parkin and MitoDsRed2 in cells co-expressing V5-tagged constructs (n = 6 wells per condition, error bars are SEM). Data from CCCP-treated conditions were analyzed by two-way ANOVA with Bonferroni's post hoc test; ***P < 0.001, versus CCCP-treated control shRNA for each construct. (C) Immunoblots of HK1 and HK2 (filled arrows) in the human frontal cortex, human midbrain and HeLa lysates with cyclophilin B (open arrows) as a loading control. (D) Effects of knockdown of both HK1 and HK2 on parkin recruitment. HeLa cells stably expressing YFP-parkin were transfected with siRNA against PINK1, HK1, HK2 or both HK1 and HK2 and were treated with 2 μm CCCP (2 h, low-glucose media) for 2 h, fixed and analyzed for the mean area of discreet YFP-parkin ‘spots’ per cell (n = 5 wells per condition, error bars are SEM). Data from CCCP-treated conditions were analyzed by one-way ANOVA with Dunnett's post hoc test; ***P < 0.001, significantly different from non-targeting control siRNA condition. (E) HeLa lines stably expressing YFP-parkin, MitoDsRed2 and either control, HK2 or PINK1 shRNA were transfected with V5-tagged LacZ, HK1 WT, HK1 KD, HK2 WT or HK2 KD constructs and treated with 2 μm CCCP (2 h, low-glucose media). Data graphed are the mean overlap area between YFP-parkin and MitoDsRed2 in cells co-expressing V5-tagged constructs (n = 3 wells per condition, error bars are SEM). Data from CCCP-treated conditions were analyzed by two-way ANOVA with Bonferroni's post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001, versus CCCP-treated control shRNA for each construct. (F) Primary cortical neurons transfected with YFP-parkin and V5-tagged LacZ, HK2 WT or HK2 KD constructs were treated with 10 μm CCCP for 6 h, fixed and labeled for TOM20 and V5. Scale bar: 20 μm. (G) Quantification of the proportion of neurons with discreet areas of mitochondria-localized YFP-parkin assessed under blinded conditions. P-values are by chi-squared test.

Article Snippet: We, therefore, considered HK2 to be a novel candidate modifier of parkin recruitment to mitochondria. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Clone # Gene Screen percent of cells with low correlation coefficient Validation percent of cells with low correlation coefficient TRCN0000037671 HK2 43.9 33.64 TRCN0000037672 HK2 14.22 13.59 TRCN0000037673 HK2 61.28 74.09 TRCN0000197099 HK2 76.53 6.13 TRCN0000196260 HK2 74.06 7.62 TRCN0000195582 HK2 81.36 2.78 TRCN0000199344 HK2 64.68 76.99 TRCN0000195340 HK2 89.95 68.93 TRCN0000037669 HK2 22.33 7.04 TRCN0000037670 HK2 22.46 5.16 TRCN0000007097 PINK1 16.14 5.85 TRCN0000007098 PINK1 50 52.86 TRCN0000007099 PINK1 70.87 78.57 TRCN0000007100 PINK1 68 82.61 TRCN0000007101 PINK1 95.19 91.43 TRCN0000196758 PINK1 17.03 14.07 TRCN0000197083 PINK1 92.04 96.72 TRCN0000199193 PINK1 88.24 91.04 TRCN0000199255 PINK1 81.42 58.54 Open in a separate window HK2 and PINK1 results from the screen and validation

Techniques: Stable Transfection, Expressing, shRNA, Transfection, Construct, Quantitation Assay, Western Blot, Labeling

Journal: Human Molecular Genetics

Article Title: Hexokinase activity is required for recruitment of parkin to depolarized mitochondria

doi: 10.1093/hmg/ddt407

Figure Lengend Snippet: High-content screen for modifiers of CCCP-induced parkin relocalization to depolarized mitochondria. (A) HeLa cells expressing YFP-parkin and MitoDsRed2 were transduced with PINK1 shRNA lentiviral particles, treated with 2 μm CCCP for 1 h and imaged on a Cellomics VTI arrayscan. Scale bar: 50 μm, insets show higher magnification of YFP-parkin. (B) Cultures were treated as in (A) using four independent shRNA clones directed against PINK1 and imaged. Bars show the percentage of cells from 200 counted for which the correlation coefficient between YFP-parkin and MitoDsRed2 was more than 2 standard deviations below the mean of CCCP-treated controls. Error bars denote the SEM, n = 3 wells per condition. One-way ANOVA with Dunnett's post hoc test; **P < 0.01; ***P < 0.001 versus CCCP-treated control. (C) Percentage of cells with low correlation coefficients in the high-content screen. Central line shows the median, the box denotes 1–3 quartiles and range bars denote 1–99 percentiles. (D) Comparison of percent cells with low correlation coefficients from the primary screen (x-axis) versus validation (y-axis). Each point represents a different PINK1 (magenta circles) or HK2 (black circles) shRNA.

Article Snippet: We, therefore, considered HK2 to be a novel candidate modifier of parkin recruitment to mitochondria. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Clone # Gene Screen percent of cells with low correlation coefficient Validation percent of cells with low correlation coefficient TRCN0000037671 HK2 43.9 33.64 TRCN0000037672 HK2 14.22 13.59 TRCN0000037673 HK2 61.28 74.09 TRCN0000197099 HK2 76.53 6.13 TRCN0000196260 HK2 74.06 7.62 TRCN0000195582 HK2 81.36 2.78 TRCN0000199344 HK2 64.68 76.99 TRCN0000195340 HK2 89.95 68.93 TRCN0000037669 HK2 22.33 7.04 TRCN0000037670 HK2 22.46 5.16 TRCN0000007097 PINK1 16.14 5.85 TRCN0000007098 PINK1 50 52.86 TRCN0000007099 PINK1 70.87 78.57 TRCN0000007100 PINK1 68 82.61 TRCN0000007101 PINK1 95.19 91.43 TRCN0000196758 PINK1 17.03 14.07 TRCN0000197083 PINK1 92.04 96.72 TRCN0000199193 PINK1 88.24 91.04 TRCN0000199255 PINK1 81.42 58.54 Open in a separate window HK2 and PINK1 results from the screen and validation

Techniques: Expressing, Transduction, shRNA, Clone Assay

Journal: Human Molecular Genetics

Article Title: Hexokinase activity is required for recruitment of parkin to depolarized mitochondria

doi: 10.1093/hmg/ddt407

Figure Lengend Snippet: HK2 deficiency decreases YFP-parkin recruitment to mitochondria. (A) Relative HK2 mRNA levels in YFP-parkin HeLa cell lines with stable expression of lentiviral shRNA constructs targeting HK2 or PINK1 normalized to β-actin. Error bars are SEM (n = 4). One-way ANOVA with Dunnett's post hoc test; **P < 0.01; ***P < 0.001 versus control shRNA. (B) Western blot of PINK1 and HK2 shRNA lines probed for HK2 (closed arrow) and β-actin (open arrow). (C) Quantitation of (B) with HK2 values normalized to β-actin relative to control shRNA; error bars are SEM (n = 3). One-way ANOVA with Dunnett's post hoc test; **P < 0.01 versus control shRNA. (D) Cell lines stably expressing YFP-parkin, and either control, PINK1 or HK2 shRNA-treated 2 μm CCCP (2 h), were fixed, stained with anti-TOM20 and imaged by confocal microscopy. Scale bar: 20 μm. (E) Stable HeLa lines expressing control, PINK1 or HK2 shRNA were treated with 2 μm CCCP (2 h in low-glucose media), fixed and imaged on the Cellomics VTI arrayscan. Bars show the mean overlap area between YFP-parkin and MitoDsRed2 structures (n = 6 wells per condition, error bars indicate the SEM). Data from CCCP-treated conditions were analyzed by one-way ANOVA with Dunnett's post hoc test; **P < 0.01; ***P < 0.001 versus CCCP-treated control shRNA. (F) Living cells labeled with Hoechst 33342 were imaged on the Cellomics VTI arrayscan every 30 min for 4 h after treatment with 2 μm CCCP. Data points are mean areas of discreet YFP-parkin ‘spots’ per cell and error bars are SEM. Data were analyzed by repeated measure two-way ANOVA with Bonferroni's post hoc test. All PINK1 and HK2 shRNA lines were significantly different from CCCP-treated control shRNA at all timepoints after 1h (P < 0.001). (G) Cell lines were treated for 24 h with 2 μm CCCP, fixed and stained with anti-TOM20. Scale bar: 20 μm. (H) Quantitation from (G), of the total TOM20 signal (n = 10 wells per condition, error bars indicate SEM). Data from CCCP-treated conditions were analyzed by one-way ANOVA with Dunnett's post hoc test, ***P < 0.001, significantly different from CCCP-treated control shRNA.

Article Snippet: We, therefore, considered HK2 to be a novel candidate modifier of parkin recruitment to mitochondria. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Clone # Gene Screen percent of cells with low correlation coefficient Validation percent of cells with low correlation coefficient TRCN0000037671 HK2 43.9 33.64 TRCN0000037672 HK2 14.22 13.59 TRCN0000037673 HK2 61.28 74.09 TRCN0000197099 HK2 76.53 6.13 TRCN0000196260 HK2 74.06 7.62 TRCN0000195582 HK2 81.36 2.78 TRCN0000199344 HK2 64.68 76.99 TRCN0000195340 HK2 89.95 68.93 TRCN0000037669 HK2 22.33 7.04 TRCN0000037670 HK2 22.46 5.16 TRCN0000007097 PINK1 16.14 5.85 TRCN0000007098 PINK1 50 52.86 TRCN0000007099 PINK1 70.87 78.57 TRCN0000007100 PINK1 68 82.61 TRCN0000007101 PINK1 95.19 91.43 TRCN0000196758 PINK1 17.03 14.07 TRCN0000197083 PINK1 92.04 96.72 TRCN0000199193 PINK1 88.24 91.04 TRCN0000199255 PINK1 81.42 58.54 Open in a separate window HK2 and PINK1 results from the screen and validation

Techniques: Expressing, shRNA, Construct, Western Blot, Quantitation Assay, Stable Transfection, Staining, Confocal Microscopy, Labeling

Journal: Human Molecular Genetics

Article Title: Hexokinase activity is required for recruitment of parkin to depolarized mitochondria

doi: 10.1093/hmg/ddt407

Figure Lengend Snippet: Parkin recruitment requires both oxidative phosphorylation and PINK1 signaling. (A) HeLa lines stably expressing YFP-parkin/MitoDsRed2 and either control, PINK1 or HK2 shRNA were treated with 2 μm CCCP and 10 μm oligomycin as indicated (2 h, low-glucose media), and the mean overlap area between YFP-parkin and MitoDsRed2 was analyzed (n = 4 wells per condition, error bars are SEM). Data from CCCP-treated conditions were analyzed by two-way ANOVA with Bonferroni's post hoc test; ***P < 0.001, versus control shRNA for each treatment condition. (B) HeLa lines stably expressing YFP-parkin/MitoDsRed2 and either control, PINK1 or HK2 shRNA were cultured in glucose or galactose complete media and treated with 2 μm CCCP (2 h, low-glucose media or galactose-containing media), and the mean overlap area between YFP-parkin and MitoDsRed2 was analyzed (n = 6 wells per condition, error bars are SEM).

Article Snippet: We, therefore, considered HK2 to be a novel candidate modifier of parkin recruitment to mitochondria. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Clone # Gene Screen percent of cells with low correlation coefficient Validation percent of cells with low correlation coefficient TRCN0000037671 HK2 43.9 33.64 TRCN0000037672 HK2 14.22 13.59 TRCN0000037673 HK2 61.28 74.09 TRCN0000197099 HK2 76.53 6.13 TRCN0000196260 HK2 74.06 7.62 TRCN0000195582 HK2 81.36 2.78 TRCN0000199344 HK2 64.68 76.99 TRCN0000195340 HK2 89.95 68.93 TRCN0000037669 HK2 22.33 7.04 TRCN0000037670 HK2 22.46 5.16 TRCN0000007097 PINK1 16.14 5.85 TRCN0000007098 PINK1 50 52.86 TRCN0000007099 PINK1 70.87 78.57 TRCN0000007100 PINK1 68 82.61 TRCN0000007101 PINK1 95.19 91.43 TRCN0000196758 PINK1 17.03 14.07 TRCN0000197083 PINK1 92.04 96.72 TRCN0000199193 PINK1 88.24 91.04 TRCN0000199255 PINK1 81.42 58.54 Open in a separate window HK2 and PINK1 results from the screen and validation

Techniques: Stable Transfection, Expressing, shRNA, Cell Culture

Journal: Human Molecular Genetics

Article Title: Hexokinase activity is required for recruitment of parkin to depolarized mitochondria

doi: 10.1093/hmg/ddt407

Figure Lengend Snippet: HK2 and PINK1 results from the screen and validation

Article Snippet: We, therefore, considered HK2 to be a novel candidate modifier of parkin recruitment to mitochondria. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Clone # Gene Screen percent of cells with low correlation coefficient Validation percent of cells with low correlation coefficient TRCN0000037671 HK2 43.9 33.64 TRCN0000037672 HK2 14.22 13.59 TRCN0000037673 HK2 61.28 74.09 TRCN0000197099 HK2 76.53 6.13 TRCN0000196260 HK2 74.06 7.62 TRCN0000195582 HK2 81.36 2.78 TRCN0000199344 HK2 64.68 76.99 TRCN0000195340 HK2 89.95 68.93 TRCN0000037669 HK2 22.33 7.04 TRCN0000037670 HK2 22.46 5.16 TRCN0000007097 PINK1 16.14 5.85 TRCN0000007098 PINK1 50 52.86 TRCN0000007099 PINK1 70.87 78.57 TRCN0000007100 PINK1 68 82.61 TRCN0000007101 PINK1 95.19 91.43 TRCN0000196758 PINK1 17.03 14.07 TRCN0000197083 PINK1 92.04 96.72 TRCN0000199193 PINK1 88.24 91.04 TRCN0000199255 PINK1 81.42 58.54 Open in a separate window HK2 and PINK1 results from the screen and validation

Techniques: