Journal: Science Advances

Article Title: Parkin regulates amino acid homeostasis at mitochondria-lysosome (M/L) contact sites in Parkinson’s disease

doi: 10.1126/sciadv.adh3347

Figure Lengend Snippet: ( A ) Time-lapse images of mitochondria-lysosome (M/L) contact tethering (yellow arrow) and untethering (white arrow) in HeLa cells expressing LAMP1-Halo and mApple-TOMM20. Scale bar, 1 μm. ( B ) Time-lapse images of mitochondria-lysosome contact tethering (yellow arrow) and untethering (white arrow) in HeLa cells expressing LAMP1-Halo, mApple-TOMM20, and eGFP-Parkin or eGFP-Parkin C431S. Scale bars, 1 μm. ( C ) Quantification of percentage of lysosomes in stable contact (> 10 s) with mitochondria in HeLa cells expressing eGFP-Parkin or eGFP-Parkin C431S [from (B)] ( n = 18 cells from three biological replicates per condition). ( D and E ) Quantification (D) and distribution (E) of minimum duration of mitochondria-lysosome contacts in HeLa cells expressing eGFP-Parkin or eGFP Parkin C431S [from (B)] ( n = 126 events from 18 cells from three biological replicates per condition). ( F ) Schematic of FRET for mitochondria-lysosome contacts using FRET donor lysosomal marker LAMP1-pmTurquoise2 and FRET acceptor mitochondrial marker mCitrine-TOMM20. ( G ) Representative FRET images for mitochondria-lysosome contacts in HeLa cells expressing LAMP1-pmTurquoise, mCitrine-TOMM20, and Halo-Parkin WT or Halo-Parkin C431S. Scale bars, 10 and 1 μm (zoom). ( H ) Quantification of FRET efficiency in HeLa cells expressing LAMP1-pmTurquoise, mCitrine-TOMM20, and Halo-Parkin WT or Halo-Parkin C431S [from (G)] ( n = 28 cells from three biological replicates per condition). Data are means ± SEM [** P < 0.01 and *** P < 0.001, unpaired two-tailed t test (C, D, and H)].

Article Snippet: The following plasmids were obtained from Addgene: mApple-TOMM20-N-10 (Addgene; 54955), mCitrine-N1 (Addgene; 54594) , and EBFP2-Lysosomes-20 (Addgene; 55246) were gifts from M. Davidson; LAMP1-mGFP (Addgene; 34831) ( ) was a gift from E. Dell-Angelica; pmTurquoise2-N1 (Addgene; 60561) ( ) was a gift from D. Gadella; Halo-KDEL (Addgene; 124316) ( ) was a gift from J. Wang; pEGFP-parkin WT (Addgene; 45875) ( ) and pEGFP-parkin C431S (Addgene; 45877) ( ) were gifts from E. Fon; EGFP-Rab7A (Addgene; 28047) ( ) was a gift from Q. Zhong; pMXs-3xHA-EGFP-OMP25 (Addgene; 83356) ( ) and pLJC5-LAMP1-RFP-3xHA (Addgene; 102932) ( ) were gifts from D. Sabatini; psPAX2 (Addgene; 12260) was a gift from D. Trono; Lamp1-RFP (Addgene; 1817) ( ) was a gift from W. Mothes; pGEX-4T-3-mR7BD (Addgene; 79149) ( ) was a gift from A. Edinger; and Halo-TOMM20-N-10 (Addgene; 123284) was a gift from K. McGowan.

Techniques: Expressing, Marker, Two Tailed Test

Journal: Science Advances

Article Title: Parkin regulates amino acid homeostasis at mitochondria-lysosome (M/L) contact sites in Parkinson’s disease

doi: 10.1126/sciadv.adh3347

Figure Lengend Snippet: ( A ) GST-RILP pulldown to measure active Rab7-GTP in HeLa cells expressing eGFP (control), eGFP-Parkin WT, or eGFP-Parkin C431S. ( B ) Quantification of active Rab7-GTP levels in HeLa cells expressing eGFP (control), eGFP-Parkin WT, or eGFP-Parkin C431S ( n = 3 biological replicates). ( C ) Pearson’s correlation coefficient of eGFP-Rab7 (WT or K38R) with BFP-lysosomes in HeLa cells coexpressing mock (control), Halo-Parkin WT, or Halo-Parkin C431S ( n = 20 cells from three biological replicates per condition). ( D ) Fluorescence recovery after photobleaching (FRAP) images of Rab7 onto LAMP1-positive vesicles from HeLa cells expressing LAMP1-RFP, eGFP-Rab7, and Halo-Parkin WT or Halo-Parkin C431S. Scale bars, 1 μm. ( E and F ) Fluorescence recovery over time (E) and quantification of FRAP recovery rate (F) of Rab7 onto LAMP1-positive vesicles in HeLa cells expressing Halo-Parkin WT or Halo-Parkin C431S [from (D)] ( n = 9 cells from three biological replicates per condition). ( G ) GST-RILP pulldown to measure active Rab7-GTP in Parkin PD and isogenic control iPSC-derived dopaminergic neurons. ( H ) Quantification of active Rab7-GTP levels in Parkin PD and isogenic control iPSC-derived dopaminergic neurons [from (G)] ( n = 3 biological replicates). ( I and J ) Representative Western blots (I) and protein quantification (J) of various mitochondria-lysosome contact site-regulating proteins in Parkin PD and isogenic control iPSC-derived dopaminergic neurons ( n = 3 biological replicates). Data are means ± SEM [* P < 0.05 and ** P < 0.01, ns, not significant; unpaired two-tailed t test (F, H, and J), one-way analysis of variance (ANOVA) with Tukey’s post hoc test (B and C)].

Article Snippet: The following plasmids were obtained from Addgene: mApple-TOMM20-N-10 (Addgene; 54955), mCitrine-N1 (Addgene; 54594) , and EBFP2-Lysosomes-20 (Addgene; 55246) were gifts from M. Davidson; LAMP1-mGFP (Addgene; 34831) ( ) was a gift from E. Dell-Angelica; pmTurquoise2-N1 (Addgene; 60561) ( ) was a gift from D. Gadella; Halo-KDEL (Addgene; 124316) ( ) was a gift from J. Wang; pEGFP-parkin WT (Addgene; 45875) ( ) and pEGFP-parkin C431S (Addgene; 45877) ( ) were gifts from E. Fon; EGFP-Rab7A (Addgene; 28047) ( ) was a gift from Q. Zhong; pMXs-3xHA-EGFP-OMP25 (Addgene; 83356) ( ) and pLJC5-LAMP1-RFP-3xHA (Addgene; 102932) ( ) were gifts from D. Sabatini; psPAX2 (Addgene; 12260) was a gift from D. Trono; Lamp1-RFP (Addgene; 1817) ( ) was a gift from W. Mothes; pGEX-4T-3-mR7BD (Addgene; 79149) ( ) was a gift from A. Edinger; and Halo-TOMM20-N-10 (Addgene; 123284) was a gift from K. McGowan.

Techniques: Expressing, Control, Fluorescence, Derivative Assay, Western Blot, Two Tailed Test