Journal: JIMD Reports

Article Title: Diurnal variability of glucose tetrasaccharide (Glc 4 ) excretion in patients with glycogen storage disease type III

doi: 10.1002/jmd2.12181

Figure Lengend Snippet: Summary of diurnal variability of Glc 4 excretion in subjects with GSD III

Article Snippet: Acquity UPLC BEH amide 2.1 × 100 mm column, VanGuard guard column, and Sep‐Pak Vac C18 cartridges were obtained from Waters (Milford, Massachusetts), Glc 4 from Toronto Research Chemicals (Toronto, Canada), glacial acetic acid, butyl‐4‐aminobenzoate, sodium cyanoborohydride from Sigma (St. Louis, MO), and methanol and acetonitrile (HPLC grade) from VWR Scientific products (Atlanta, Georgia).

Techniques:

Journal: JIMD Reports

Article Title: Diurnal variability of glucose tetrasaccharide (Glc 4 ) excretion in patients with glycogen storage disease type III

doi: 10.1002/jmd2.12181

Figure Lengend Snippet: Comparison of the degree and variability of Glc 4 excretion with uncooked cornstarch dose. A, Glc 4 concentrations in 24‐hour urine relative to total cornstarch dose. No significant correlation was observed (Pearson R = −.349, P = .15, n = 18 patients). B, Variability (CV%) in Glc 4 excretion in 24‐hour urine relative to cornstarch dose. No significant correlation was observed (Pearson R = −.278, P = .15, n = 28 collections by 15 subjects)

Article Snippet: Acquity UPLC BEH amide 2.1 × 100 mm column, VanGuard guard column, and Sep‐Pak Vac C18 cartridges were obtained from Waters (Milford, Massachusetts), Glc 4 from Toronto Research Chemicals (Toronto, Canada), glacial acetic acid, butyl‐4‐aminobenzoate, sodium cyanoborohydride from Sigma (St. Louis, MO), and methanol and acetonitrile (HPLC grade) from VWR Scientific products (Atlanta, Georgia).

Techniques:

Journal: Experimental Animals

Article Title: Abietic acid attenuates sepsis-induced lung injury by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway to inhibit M1 macrophage polarization

doi: 10.1538/expanim.22-0018

Figure Lengend Snippet: Abietic acid inhibited M1 macrophage polarization in BALF of sepsis mice. (A) Representative flow scatter plot of M1 macrophages (F4/80+CD86+ cells) in BALF detected by flow cytometry. (B) Representative flow scatter plot of M2 macrophages (F4/80+CD163+ cells) in BALF detected by flow cytometry. (C) Statistical comparison of the proportion of M1 macrophages in BALF from 7 mice per group. (D) Statistical comparison of the proportion of M2 macrophages in BALF from 7 mice per group. Statistical comparison of the proportion of M1/M2 macrophages ratio in BALF from 7 mice per group. Data from 7 mice for each group are plotted as (mean ± SD) as a column chart, and P values were calculated by analysis of variance. *** P <0.001 vs. Sham group, ns P >0.05, # P <0.05, ## P <0.01 and ### P <0.001 vs. CLP group.

Article Snippet: Next, cells in BALF were stained with anti-CD86-FITC (99879, Cell Signaling Technology, Boston, MA, USA), anti-CD163-APC (MA5-17717, Invitrogen, Grand Island, NY, USA) and anti-F4/80-PE (80380, Cell Signaling Technology) for 30 min in the dark at 4°C.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: The lysosomal chloride-proton exchanger CLC7 functions in melanosomes as a negative regulator of human pigmentation

doi: 10.1101/2021.02.05.430016

Figure Lengend Snippet: A. Representative bright field images of HEMs expressing CLC7− and OSTM1-targeting shRNA show increased pigmentation compared to HEMs expressing control non-targeting (NT) shRNA. Scale bar: 20 μm. B. CLC7 mRNA levels (green bars) were reduced by 84% in HEMs expressing CLC7 shRNA (P = 0.0027) and slightly increased in cells expressing OSTM1 shRNA (P = 0.1367) compared to control NT shRNA cells. OSTM1 mRNA levels (blue bars) were reduced by 81% in OSTM1 shRNA expressing HEMs (P = 0.0009) and by 74% in HEMs expressing CLC7 shRNA (P = 0.0256) compared to NT shRNA cells. P-values derived from paired two-sample t-tests. ± SEM; n ≥ 3 independent experiments per condition. C. Compared to HEMs expressing NT shRNA, CLC7 protein levels were decreased by 94% in CLC7 shRNA cells (P = 0.0004) and by 59% in OSTM1 shRNA cells (P = 0.0257) Inset: Representative immunoblot showing attenuated CLC7 protein levels in HEMs expressing CLC7 or OSTM1 shRNA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. P-values derived from paired two-sample t-tests. ± SEM; n = 3 independent experiments. D. Total cellular melanin concentration is increased 2.1-fold in HEMs expressing CLC7-targeting shRNA (green bar; P = 0.0161) and 3.0-fold in OSTM1-targeting shRNA expressing HEMs (blue bar; P = 0.0178), compared to NT shRNA cells. P-values derived from paired two-sample t-tests. ± SEM; n = 4 independent experiments. E. Total cellular concentration of eumelanin (measured as PTCA, see Methods; brown bars) is increased in HEMs expressing CLC7- or OSTM1-targeting compared to NT shRNA (P = 0.0001 and 0.0004, respectively). The total cellular concentration of pheomelanin (measured as BZ-PM + BT-PM, see Methods; orange bars) is increased by 76% in CLC7 shRNA cells (P = 0.0004) and decreased by 26% in OSTM1 shRNA cells (P = 0.0006). P-values derived from paired two-sample t-tests. ± SEM; n = 3 independent experiments.

Article Snippet: For pcDNA4/TO-OSTM1-Myc, the OSTM1-Myc insert from pCMV6-OSTM1-MYC-FLAG (RC209871, Origene) was amplified by PCR using forward primers (OSTM1: 5’-CATCATCTCGAGATGGAGCCGGGCCCGAC) and reverse primers (Myc-STOP: 5’-CATCATTCTAGACTACAGATCCTCTTCTGAGATGAGTTTCTGCTC) containing the XhoI/XbaI restriction sites at the 5’ or 3’ ends, respectively.

Techniques: Expressing, shRNA, Derivative Assay, Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: The lysosomal chloride-proton exchanger CLC7 functions in melanosomes as a negative regulator of human pigmentation

doi: 10.1101/2021.02.05.430016

Figure Lengend Snippet: A. Representative confocal images of HEMs immunostained with antibodies to lysosome-associated membrane protein 1 (LAMP1; top) and tyrosinase related protein 1 (TYRP1; bottom) to visualize lysosomes and melanosomes, respectively. Immunostaining reveals CLC7 colocalization with LAMP1- and TYRP1-stained organelles and darkly pigmented melanosomes observed by bright field (BF) microscopy. Inset: Magnified view of areas in the white boxes. Arrows indicate organelles with endogenous CLC7. Scale bar: 10 μm. B. Quantification of images as in A , showing reciprocal overlap between endogenous CLC7 and LAMP1 (top) or TYRP1 (bottom) in HEMs. Endogenous CLC7 localizes to 49% and 45% of all LAMP1- and TYRP1-stained organelles, respectively. LAMP1- and TYRP1-stained organelles colocalize with 51% and 42% of total endogenous CLC7, respectively. ± SEM; n = 4 independent experiments. C. Representative confocal images of HEMs transiently transfected with GFP-CLC7 and OSTM1-Myc and immunostained with antibodies to LAMP1 (top) and TYRP1 (bottom). Similar to endogenous CLC7, overexpressed GFP-CLC7 localizes to LAMP1- and TYRP1-stained organelles. Inset: Magnified view of areas in the white boxes. Arrows indicate stained organelles with GFP-CLC7. Scale bar: 10 μm. D. Quantification of images as in C showing reciprocal overlap between overexpressed GFP-CLC7 and LAMP1 (top) or TYRP1 (bottom) in HEMs. GFP-CLC7 localizes to 36% and 31% of all LAMP1- and TYRP1-stained organelles, respectively. LAMP1- and TYRP1-stained organelles colocalize with 43% and 54% of total GFP-CLC7, respectively. ± SEM; n = 4 independent experiments.

Article Snippet: For pcDNA4/TO-OSTM1-Myc, the OSTM1-Myc insert from pCMV6-OSTM1-MYC-FLAG (RC209871, Origene) was amplified by PCR using forward primers (OSTM1: 5’-CATCATCTCGAGATGGAGCCGGGCCCGAC) and reverse primers (Myc-STOP: 5’-CATCATTCTAGACTACAGATCCTCTTCTGAGATGAGTTTCTGCTC) containing the XhoI/XbaI restriction sites at the 5’ or 3’ ends, respectively.

Techniques: Immunostaining, Staining, Microscopy, Transfection

Journal: bioRxiv

Article Title: The lysosomal chloride-proton exchanger CLC7 functions in melanosomes as a negative regulator of human pigmentation

doi: 10.1101/2021.02.05.430016

Figure Lengend Snippet: A. Schematic representation of OCA2-mediated regulation of melanosome pH and melanin synthesis. Cl − efflux via OCA2 decreases vacuolar ATPase (V-ATPase) activity resulting in elevated tyrosinase (TYR) activity and melanin synthesis. B. Representative confocal fluorescence micrographs of HEMs and MNT1 cells transiently transfected with GFP-CLC7, OSTM1-Myc, visualized by anti-Myc (α-Myc) immunostaining, and mCh-OCA2. Inset: Magnified view of the area in white boxe, with arrows indicating organelles coexpressing GFP-CLC7, OSTM1-Myc, and mCh-OCA2. Scale bar: 10 μm. C. Quantification of images as in B shows overlap between overexpressed GFP-CLC7 and OSTM1-Myc or mCh-OCA2 in HEMs (top) and MNT1 cells (bottom). In both HEMs and MNT1 cells, ≥64% and ≥62% of total GFP-CLC7 localizes to organelles expressing OSTM1-Myc or mCh-OCA2, respectively. At least 60% of organelles with mCh-OCA2 in HEMs and MNT1 cells colocalize with GFP-CLC7. ± SEM; n ≥ 3 independent experiments. D. Total cellular melanin concentration in MNT1 cells transiently expressing for five days mCh-OCA2 alone or together with GFP-CLC7 and OSTM1-Myc. Compared to MNT1 cells expressing CLC7 and OSTM1, melanin content is increased 2.8-fold in cells expressing OCA2 alone (P = 0.0867) and 1.7-fold when OCA2 was expressed together with CLC7 and OSTM1 (P = 0.2163). P-values derived from unpaired two-sample t-tests. ± SEM; n ≥ 3 independent experiments.

Article Snippet: For pcDNA4/TO-OSTM1-Myc, the OSTM1-Myc insert from pCMV6-OSTM1-MYC-FLAG (RC209871, Origene) was amplified by PCR using forward primers (OSTM1: 5’-CATCATCTCGAGATGGAGCCGGGCCCGAC) and reverse primers (Myc-STOP: 5’-CATCATTCTAGACTACAGATCCTCTTCTGAGATGAGTTTCTGCTC) containing the XhoI/XbaI restriction sites at the 5’ or 3’ ends, respectively.

Techniques: Activity Assay, Fluorescence, Transfection, Immunostaining, Expressing, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: The lysosomal chloride-proton exchanger CLC7 functions in melanosomes as a negative regulator of human pigmentation

doi: 10.1101/2021.02.05.430016

Figure Lengend Snippet: A. MNT1 cells expressing iRFP-CLC7-Y715C and OSTM1-Myc have enlarged cytosolic vacuoles, as observed by bright field (BF) microscopy. OSTM1-Myc was detected by anti-Myc immunostaining. Scale bar: 10 μm. B. Wide-field micrographs of MNT1 cells expressing mCh, iRFP-CLC7-Y715C, and OSTM1-Myc and incubated with LysoSensor show that the enlarged cytosolic vacuoles that exclude mCh accumulate LysoSensor, visualized using its yellow fluorescent emission (Lyso-W1). Scale bar: 10 μm. C. Representative widefield fluorescence images of LysoSensor-stained MNT1 cells transiently coexpressing OSTM1-Myc with either iRFP-CLC7-Y715C (top) or iRFP-CLC7-WT (bottom). LysoSensor dual fluorescence emission Lyso-W1 (yellow) and Lyso-W2 (blue), and the ratio W1/W2 are shown in pseudocolor, with more yellow W1/W2 indicating increased organelle acidity, as shown by the pH scale. Scale bar: 10 μm. D. Average cellular organelle area (in μm 2 ) obtained as individual LysoSensor-stained organelles area the averaged for individual MNT1 cells expressing the indicated proteins. The average organelle area per MNT1 cell transiently expressing OSTM1-Myc with iRFP-CLC7-WT or iRFP-CLC7-Y715C is (0.77 ± 0.04) μm 2 and (3.08 ± 0.47) μm 2 , respectively. (P < 0.0001, Mann-Whitney test). Bar graphs represent mean ± SEM. E. Surface area of individual LysoSensor-stained organelles from MNT1 cells expressing OSTM1-Myc together with iRFP-CLC7-WT (grey circles) or iRFP-CLC7-Y715C (green circles). The average organelle area in cells with CLC7-Y715C is 3.8-fold higher compared to CLC7-WT cells (P < 0.0001, Mann-Whitney test), indicating that CLC7-Y715C overexpression results in increased organelle size. Black lines represent mean ± SEM. F. Frequency distribution of organelle area from values obtained in E . CLC7-Y715C expression shifts the organelle area distribution to higher values, indicating increased organelle size in CLC7-Y715C cells compared to CLC7-WT cells (P < 0.0001, Kolmogorov-Smirnov test). G. Average cellular ratio of the two emission intensities of LysoSensor (I W1 /I W2 ) obtained as the average of LysoSensor-stained organelles from individual MNT1 cells expressing the indicated proteins. The average organelle IW1/IW2 per MNT1 cell transiently expressing OSTM1-Myc with iRFP-CLC7-WT or iRFP-CLC7-Y715C is 0.32 ± 0.02 and 0.88 ± 0.10, respectively (± SEM; P < 0.0001, Mann-Whitney test). A higher I W1 /I W2 indicates increased organelle acidity as shown by the pH scale (right). Box plots show the data medians, 25 th and 75 th percentiles, and extrema. H. I W1 /I W2 values of individual LysoSensor-containing organelles from MNT1 cells overexpressing OSTM1-Myc with iRFP-CLC7-WT (grey circles) or iRFP-CLC7-Y715C (green circles). The average organelle I W1 /I W2 in cells with CLC7-Y715C is increased 2.8-fold compared to CLC7-WT cells, indicating that CLC7-Y715C increases organelle acidity (P < 0.0001, Mann-Whitney test). Black lines represent mean ± SEM. I. Frequency distribution of organelle IW1/IW2 from values obtained in H . CLC7-Y715C expression shifts the organelle I W1 /I W2 distribution to higher values, indicating increased organelle acidity in CLC7-Y715C cells compared to CLC7-WT cells (P < 0.0001, Kolmogorov-Smirnov test). Organelle I W1 /I W2 measurements in G-I were paired with organelle area measurements in D-F . ≥ 367 organelles/cell from ≥ 8 cells were analyzed; n = 5 independent experiments.

Article Snippet: For pcDNA4/TO-OSTM1-Myc, the OSTM1-Myc insert from pCMV6-OSTM1-MYC-FLAG (RC209871, Origene) was amplified by PCR using forward primers (OSTM1: 5’-CATCATCTCGAGATGGAGCCGGGCCCGAC) and reverse primers (Myc-STOP: 5’-CATCATTCTAGACTACAGATCCTCTTCTGAGATGAGTTTCTGCTC) containing the XhoI/XbaI restriction sites at the 5’ or 3’ ends, respectively.

Techniques: Expressing, Microscopy, Immunostaining, Incubation, Fluorescence, Staining, MANN-WHITNEY, Over Expression

Journal: bioRxiv

Article Title: The lysosomal chloride-proton exchanger CLC7 functions in melanosomes as a negative regulator of human pigmentation

doi: 10.1101/2021.02.05.430016

Figure Lengend Snippet: A. Total cellular melanin concentration of MNT1 cells transiently expressing iRFP-CLC7-Y715C and OSTM1-Myc (orange bar) is decreased 2.3-fold compared to mock-transfected control cells (left brown bar, P = 0.0166, One-way ANOVA with Tukey’s multiple comparisons test), and is restored to control levels by ectopic expression of WT mCh-OCA2 (right brown bar; P = 0.9994, One-way ANOVA with Tukey’s multiple comparisons test). Bars represent mean values ± SEM; n ≥ 3 independent experiments. B. Representative widefield fluorescence images of LysoSensor-stained MNT1 cells transiently expressing iRFP-CLC7-Y715C and OSTM1-Myc with either mCh-OCA2-V443I (top) or mCh-OCA2-WT (bottom). LysoSensor dual fluorescence emission Lyso-W1 (yellow) and Lyso-W2 (blue) and the ratio W1/W2 are shown in pseudocolor, with more yellow W1/W2 indicating increased organelle acidity, as shown by the pH scale. Scale bar: 10 μm. C. Average cellular organelle area (in μm 2 ) obtained as the average of LysoSensor-stained organelles from individual MNT1 cells expressing the proteins indicated below. The average organelle area per MNT1 cell expressing iRFP-CLC7-Y715C and OSTM1-Myc alone (3.08 ± 0.70 μm 2 ) was not significantly changed by coexpression with mCherry-OCA2-V443I (3.03 ± 0.52 μm 2 ), while coexpression of mCherry-OCA2-WT decreased the average organelle area to 1.50 ± 0.28 μm 2 (P = 0.0879, One-way ANOVA with Tukey’s multiple comparisons test). Each dot represents the average organelle area of one cell. Bars represent the mean of all cells per condition ± SEM. D. Area of individual LysoSensor-stained organelles from MNT1 cells overexpressing iRFP-CLC7-Y715C and OSTM1-Myc (grey circles) with mCherry-OCA2-WT (green circles) or mCherry-OCA2-V443I (purple circles), as indicated. Compared to control cells expressing CLC7-Y715C and OSTM1 alone or with OCA2-V443I, the average organelle area is decreased by ≥ 2.0-fold when OCA2-WT is coexpressed (P < 0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test). Black lines represent the mean ± SEM. E. Frequency distribution of organelle area values obtained in D . Compared to control cells expressing CLC7-Y715C and OSTM1 with OCA2-V443I, coexpression of OCA2-WT shifts the organelle area distribution to lower values, indicating reduced organelle size (P < 0.0001, Kolmogorov-Smirnov test). F. Average cellular ratio (I W1 /I W2 ) of the fluorescence intensity of Lyso-W1 (I W1 ) and Lyso-W2 (I W2 ) of individual LysoSensor-stained organelles from individual MNT1 cells expressing the indicated proteins. The average organelle I W1 /I W2 per cell in MNT1 overexpressing iRFP-CLC7-Y715C and OSTM1-Myc alone or together with mCherry-OCA2-V443I were similar, 0.64 ± 0.07 and 0.72 ± 0.11, respectively, while coexpression of mCh-OCA2-WT resulted in lower IW1/IW2 values (0.37 ± 0.06), indicating less acidic organelles. (P = 0.0180; One-way ANOVA with Tukey’s multiple comparisons test). Dots represent individual cells, box plots show the data medians, 25 th and 75 th percentiles, and extrema. G. I W1 /I W2 values of individual LysoSensor-stained organelles from MNT1 cells overexpressing iRFP-CLC7-Y715C and OSTM1-Myc (grey circles) with mCherry-OCA2-WT (green circles) or mCherry-OCA2-V443I (purple circles). Compared to control cells expressing CLC7-Y715C and OSTM1 with or without OCA2-V443I, the average organelle I W1 /I W2 is decreased by ≥ 1.6-fold in cells rescued with OCA2-WT (P < 0.0001; Kruskal-Wallis test with Dunn’s multiple comparisons test). Mean ± SEM H. Frequency distribution of organelle IW1/IW2 values shown in G . Compared to control cells expressing CLC7-Y715C and OSTM1 with OCA2-V443I, OCA2-WT coexpression shifts the organelle IW1/IW2 distribution to lower values, indicating reduced organelle acidity (P < 0.0001, Kolmogorov-Smirnov test). Organelle I W1 /I W2 measurements in F-H were paired with organelle area measurements in C-E . Area and I W1 /I W2 measurements were acquired from ≥ 253 organelles per cell from at least 9 cells; n = 3 independent experiments.

Article Snippet: For pcDNA4/TO-OSTM1-Myc, the OSTM1-Myc insert from pCMV6-OSTM1-MYC-FLAG (RC209871, Origene) was amplified by PCR using forward primers (OSTM1: 5’-CATCATCTCGAGATGGAGCCGGGCCCGAC) and reverse primers (Myc-STOP: 5’-CATCATTCTAGACTACAGATCCTCTTCTGAGATGAGTTTCTGCTC) containing the XhoI/XbaI restriction sites at the 5’ or 3’ ends, respectively.

Techniques: Concentration Assay, Expressing, Transfection, Fluorescence, Staining

Journal: bioRxiv

Article Title: The lysosomal chloride-proton exchanger CLC7 functions in melanosomes as a negative regulator of human pigmentation

doi: 10.1101/2021.02.05.430016

Figure Lengend Snippet: A. Representative widefield fluorescence images of LysoSensor-stained HeLa cells expressing iRFP-CLC7-Y715C and OSTM1-Myc with mCh-OCA2-V443I (top) or mCh-OCA2-WT (bottom). LysoSensor dual fluorescence emission Lyso-W1 (yellow) and Lyso-W2 (blue) and the ratio W1/W2 are in pseudocolor, with more yellow W1/W2 indicating higher acidity as shown by the pH scale. Scale bar: 10 μm. B. Average organelle area (in μm 2 ) per cell obtained as the average of LysoSensor-stained organelles from individual HeLa cells expressing the indicated proteins. The average organelle area per HeLa cell overexpressing iRFP-CLC7-Y715C and OSTM1-Myc with mCherry-OCA2-WT or mCherry-OCA2-V443I is 1.04 ± 0.23 μm 2 and 4.41 ± 0.77 μm 2 , respectively (± SEM; P = 0.0008, Kruskal-Wallis test with Dunn’s multiple comparisons test). Each dot represents one cell. Bars represent the mean of all cells per condition ± SEM. C. Area values of individual LysoSensor-stained organelles from HeLa cells overexpressing iRFP-CLC7-Y715C and OSTM1-Myc (grey circles) with mCh-OCA2-WT (green circles) or mCh-OCA2-V443I (purple circles). Compared to control cells expressing CLC7-Y715C and OSTM1 with or without OCA2-V443I, the average organelle area is decreased by ≥ 3.3-fold in cells rescued with OCA2-WT (P < 0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test). Black lines represent mean ± SEM. D. Frequency distribution of organelle area values in C . Compared to control cells expressing CLC7-Y715C and OSTM1 with OCA2-V443I, OCA2-WT coexpression shifts the organelle area distribution to lower values, indicating that functional OCA2 rescues organelle size (P < 0.0001, Kolmogorov-Smirnov test). E. Average cellular IW1/IW2 obtained as the average of LysoSensor-stained organelles from individual HeLa cells expressing the indicated proteins. The average organelle IW1/IW2 per HeLa cell overexpressing iRFP-CLC7-Y715C and OSTM1-Myc with mCh-OCA2-WT or mCh-OCA2-V443I is 0.56 ± 0.13 and 1.81 ± 0.14, respectively (± SEM; P = 0.0049, One-way ANOVA with Tukey’s multiple comparisons test). Dots represent individual cells. Box plots show the data medians, 25 th and 75 th percentiles, and extrema. F. I W1 /I W2 values of individual LysoSensor-stained organelles from HeLa cells overexpressing iRFP-CLC7-Y715C and OSTM1-Myc (grey circles) with mCh-OCA2-WT (green circles) or mCh-OCA2-V443I (purple circles). Compared to control cells expressing CLC7-Y715C and OSTM1 with or without OCA2-V443I, the average organelle I W1 /I W2 is ≥ 1.7-fold lower in cells rescued with OCA2-WT (P < 0.0001; Kruskal-Wallis test with Dunn’s multiple comparisons test). Black lines represent mean ± SEM. G. Frequency distribution of organelle I W1 /I W2 values in F . Compared to control cells expressing CLC7-Y715C and OSTM1 with OCA2-V443I, OCA2-WT coexpression shifts the organelle IW1/IW2 distribution to lower values, indicating reduced organelle acidity (P < 0.0001, Kolmogorov-Smirnov test). Organelle I W1 /I W2 measurements in E-G were paired with organelle area measurements in B-D acquired from ≥ 281 organelles from at least 8 cells per condition; n = 3 independent experiments.

Article Snippet: For pcDNA4/TO-OSTM1-Myc, the OSTM1-Myc insert from pCMV6-OSTM1-MYC-FLAG (RC209871, Origene) was amplified by PCR using forward primers (OSTM1: 5’-CATCATCTCGAGATGGAGCCGGGCCCGAC) and reverse primers (Myc-STOP: 5’-CATCATTCTAGACTACAGATCCTCTTCTGAGATGAGTTTCTGCTC) containing the XhoI/XbaI restriction sites at the 5’ or 3’ ends, respectively.

Techniques: Fluorescence, Staining, Expressing, Functional Assay

Journal: bioRxiv

Article Title: The lysosomal chloride-proton exchanger CLC7 functions in melanosomes as a negative regulator of human pigmentation

doi: 10.1101/2021.02.05.430016

Figure Lengend Snippet: A. Melanosomes are organelles in which melanin synthesis occurs, a process mediated by the main melanogenic enzyme TYR, whose catalytic activity is pH-dependent and optimal at near-neutral pH. The melanosomal lumen is acidified by the vesicular ATPase (V-ATPase). OCA2, a melanosomal chloride Cl − channel, conducts Cl − out of melanosomes, generating a surplus of luminal positive charges that decrease the activity of the electrogenic V-ATPase. Such action increases melanosome pH and TYR activity. CLC7/OSTM1 also reside in melanosomes and function together as a 2Cl − /1H + antiporter, pumping Cl − into the lumen, which increases the V-ATPase driving force and acidifies the melanosome lumen. Under physiological conditions (middle), CLC7/OSTM1 opposes the effect of OCA2 on V-ATPase and luminal pH. With loss of CLC7/OSTM1 expression (right), OCA2 activity leads to higher luminal pH and TYR activity that leads to increased melanin synthesis. Melanosomes expressing the gain-of-function CLC7-Y715C/OSTM1 mutant (left) have a more acidic luminal pH, which lowers TYR activity and decreases melanin levels. B. In lysosomes, CLC7/OSTM1 also increases the driving force of V-ATPase to acidify the lumen (left). Expression of the gain-of-function CLC7-Y715C/OSTM1 mutant (middle) further enhances the activity of V-ATPase, resulting in hyperacidified lysosomes and impaired organelle function, as observed in the patients carrying this mutation. The effect of CLC7-Y715C/OSTM1 on lysosomal pH can be restored if OCA2 is also expressed in these lysosomes (right) to counteract the increased V-ATPase activity and hyperacidic lysosome pH.

Article Snippet: For pcDNA4/TO-OSTM1-Myc, the OSTM1-Myc insert from pCMV6-OSTM1-MYC-FLAG (RC209871, Origene) was amplified by PCR using forward primers (OSTM1: 5’-CATCATCTCGAGATGGAGCCGGGCCCGAC) and reverse primers (Myc-STOP: 5’-CATCATTCTAGACTACAGATCCTCTTCTGAGATGAGTTTCTGCTC) containing the XhoI/XbaI restriction sites at the 5’ or 3’ ends, respectively.

Techniques: Activity Assay, Expressing, Mutagenesis