gfp rab7 q67l  (Addgene inc)

Journal: Nature Communications

Article Title: Endosomal trafficking participates in lipid droplet catabolism to maintain lipid homeostasis

doi: 10.1038/s41467-025-57038-8

Figure Lengend Snippet: A The knockdown efficiency of Rab7a in A549 cells was assessed by western blot analysis ( p < 0.0001). B Control or Rab7a-knockdown A549 cells were fixed by 4% PFA, and then stained by Bodipy 493/503 (1 μM) and DAPI (1 μg/mL) for 30 min. The number and size of LDs, and fluorescence intensity of Bodipy 493/503 were quantified by ImageJ ( n = 30 for all figures; p < 0.0001 for number of LDs and relative Bodipy fluorescence, p = 0.3763 for average size of LDs). C Triglyceride level in control or Rab7a-knockdown A549 cells was determined ( p < 0.0001). D A549 cells were transiently transfected with GFP-Rab7a, GFP-Rab7a-T22N, or GFP-Rab7a-Q67L, and then stained with Nile red (1 μM) for 30 min. The number and size of LDs, and fluorescence intensity of Nile Red were quantified by ImageJ ( n = 13 for all figures; for number of LDs, p = 0.0097 for Rab7a T22N, p < 0.0001 for Rab7a Q67L; for average size of LDs, p = 0.4779 for Rab7a T22N, p < 0.0001 for Rab7a Q67L; and for relative Bodipy fluorescence, p < 0.0001 for both Rab7a T22N and Rab7a Q67L). Images were captured by a Zeiss 880 microscope with a 63x objective lens. The scale bar is 5 μm. The graphs represented data from three independent experiments. The statistical significance of differences was determined by using the unpaired two-tailed student’s t test, and data quantifications were expressed as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001; ns: no significance.

Article Snippet: pEGFP-C1-ADRP (addgene, #87161), mcherry-ACSL3 (addgene, #87158), GFP-Rab5 (addgene, #174454), mcherry-Rab5 (addgene, #55126), mcherry-Rab5 S23N, GFP-Rab7 (addgene, #61803), mcherry-Rab7 (addgene, #55127), GFP-Rab7 T22N (addgene, #28048), GFP-Rab7 Q67L (addgene, #28049), pCDNA3.1-Twinstrep Rab5 (Elife.

Techniques: Knockdown, Western Blot, Control, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test

Journal: eLife

Article Title: Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function

doi: 10.7554/eLife.100928

Figure Lengend Snippet: ( A, B ) High content microscopy (HCM) imaging and quantification of LC3 response (puncta/cell of endogenous LC3 immunofluorescent profiles) in HeLa WT , and HeLa VPS35-KO cells in response to lysosomal damage by LLOMe (1 mM, 30 and 60 min). Scale bar, 20 μm. Data, means ± SE ( n = 5), one-way ANOVA with Tukey’s multiple comparisons. ( C–E ) HCM quantification of GLUT1 response (puncta/cell of endogenous GLUT1) ( D ) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining) ( E ) in Huh7 WT treated with or without Monensin (100 µM), LLOMe (100 µM), and Bafilomycin A1 (100 nM) for 45 min. Scale bar, 20 μm. Data, means ± SE ( n = 5), one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell ( F, G ) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining), ( H ) phenotype monitored by HCM quantification in Huh7 WT cells transfected with GFP (control) or GFP-Rab7 WT , GFP-Rab7 Q67L , and GFP-Rab7 T22N , expressing plasmids. Scale bar, 20 μm. Data, means ± SE ( n = 3); one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell ( I, J ) by HCM quantification in Huh7 WT and ATG16L1-KO cells complemented with Flag (control), Flag-ATG16L1 FL , or Flag-ATG16L1 E230 , expressing plasmids. Scale bar, 20 μm. Data, means ± SE ( n = 5); one-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line/condition. Figure 7—source data 1. Numerical values for quantification in graphs.

Article Snippet: Addgene clones were: eGFP-Rab7 WT (Addgene, #12605), eGFP-Rab7 Q67L (Addgene, #28049), and eGFP-Rab7 T22N (Addgene, #28049).

Techniques: Microscopy, Imaging, Immunostaining, Transfection, Control, Expressing

Journal: eLife

Article Title: Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function

doi: 10.7554/eLife.100928

Figure Lengend Snippet: ( A, B ) High content microscopy (HCM) imaging and quantification of LC3 response (puncta/cell of endogenous LC3 immunofluorescent profiles) in HeLa WT , and HeLa VPS35-KO cells in response to lysosomal damage by LLOMe (1 mM, 30 and 60 min). Scale bar, 20 μm. Data, means ± SE ( n = 5), one-way ANOVA with Tukey’s multiple comparisons. ( C–E ) HCM quantification of GLUT1 response (puncta/cell of endogenous GLUT1) ( D ) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining) ( E ) in Huh7 WT treated with or without Monensin (100 µM), LLOMe (100 µM), and Bafilomycin A1 (100 nM) for 45 min. Scale bar, 20 μm. Data, means ± SE ( n = 5), one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell ( F, G ) and its localization to endolysosomal compartments (% of LAMP1 profiles positive for GLUT1 immunostaining), ( H ) phenotype monitored by HCM quantification in Huh7 WT cells transfected with GFP (control) or GFP-Rab7 WT , GFP-Rab7 Q67L , and GFP-Rab7 T22N , expressing plasmids. Scale bar, 20 μm. Data, means ± SE ( n = 3); one-way ANOVA with Tukey’s multiple comparisons. Analysis of GLUT1 puncta/cell ( I, J ) by HCM quantification in Huh7 WT and ATG16L1-KO cells complemented with Flag (control), Flag-ATG16L1 FL , or Flag-ATG16L1 E230 , expressing plasmids. Scale bar, 20 μm. Data, means ± SE ( n = 5); one-way ANOVA with Tukey’s multiple comparisons. HCM images in all relevant panels, examples from a bank of unbiased operator-independent machine-collected and algorithm-processed fields containing a minimum of 500 primary objects/cells per well (5 wells minimum per 96-well plate; 3 plates minimum), per cell line/condition. Figure 7—source data 1. Numerical values for quantification in graphs.

Article Snippet: Addgene clones were: eGFP-Rab7 WT (Addgene, #12605), eGFP-Rab7 Q67L (Addgene, #28049), and eGFP-Rab7 T22N (Addgene, #28049).

Techniques: Microscopy, Imaging, Immunostaining, Transfection, Control, Expressing

Journal: Nature Communications

Article Title: ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

doi: 10.1038/s41467-021-25621-4

Figure Lengend Snippet: a Representative image of Rab7 positive lysosomes-mitochondria contacts that are marked by the ER in HeLa cells expressing mRFP-Rab7, GFP-KDEL, and mito-BFP. White arrows indicate the lysosome-mitochondria contacts. Line-scan analysis of relative fluorescence intensities (left to right) from the red line shown in merge. Scale bar: 1 µm. b Quantification of the percentage of lysosome-mitochondria contacts marked by the ER in HeLa cells expressing either a wild-type Rab7 or a control vector, with mito-BFP, GFP-KDEL, and Lamp1-mCherry. The graphs show the mean ± SEM, cells from three independent experiments. Two-sided unpaired t -test. c Normalized Rab7 mRNA levels in cells transfected by a siRNA targeting specifically Rab7 or a control siRNA measured by RT-qPCR. The graphs show the mean ± SEM, cells from two independent experiments. d Quantification of the percentage of lysosome-mitochondria contacts marked by the ER when Rab7 expression level was downregulated by a siRNA targeting Rab7 compared to a control siRNA in cells expressing Lamp1-mCherry, mito-BFP, and GFP-KDEL. The graphs show the mean ± SEM, cells from three independent experiments. Two-sided unpaired t -test. e Representative images of HeLa cells expressing the wild-type (WT), constitutively active (Q67L), or dominant negative (T22N) mRFP-Rab7. Note that the dominant negative Rab7 (T22N) does not localize to lysosomes but is cytosolic. Scale bar: 10 µm. f Quantification of the percentage of lysosome-mitochondria contacts that are marked by the ER in HeLa cells expressing either the 3HA-Rab7 or its mutants with Lamp1-mCherry, mito-BFP, and GFP-KDEL. The graph shows the mean ± SEM, Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test.

Article Snippet: The following constructs were made for this study: 3HA-Rab7, 3HA-Rab7, 3HA-Rab7 Q67L and 3HA-Rab7 T22N were generated by subcloning Rab7, Rab7 Q67L and Rab7 T22N into pcDNA3.1-3xHA-C1; mApple-Drp1 was made by replacing GFP by mApple (from mApple-TOM20) in the GFP-Drp1 plasmid; CFP-GAI-Sac1 was generated by subcloning Sac1 from GFP-ORPSAC1 into CFP-GAI(1-92) (gift from Takanari Inoue, Addgene # 37307) and CFP-GAI-Sac1 C392S was generated from CFP-GAI-Sac1 via site directed mutagenesis; GFP-ORPOSBP was made by replacing amino acid 334 to 950 of ORP1L from GFP-ORP1L by amino acid 184 to 809 of OSBP from mCherry-OSBP (gift from John Brumell, Hospital for Sick Children, Toronto); GFP-ORPOSBP HH/AA was made from GFP-ORPOSBP using site directed mutagenesis; iRFP-GID1-MFF was made by replacing Rab7 from iRFP-GID1-Rab7 by MFF from GFP-MFF (gift from Glia Voeltz, Addgene #49153); GFP-Sac1-MFF and GFP-Sac1 C392S-MFF were made by introducing wild-type and C392S mutant Sac1, from CFP-GAI-Sac1 and CFP-GAI-Sac1 C392S respectively, between GFP and MFF in the GFP-MFF plasmid.

Techniques: Expressing, Fluorescence, Control, Plasmid Preparation, Transfection, Quantitative RT-PCR, Dominant Negative Mutation, Comparison

Journal: Nature Communications

Article Title: ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

doi: 10.1038/s41467-021-25621-4

Figure Lengend Snippet: a Representative image of a cell expressing GFP-ORP1L and mApple-TOM20 acquired by SIM microscopy. White arrows show ORP1L positive lysosomes-mitochondria contacts. Scale bars: 10 µm and 1 µm (inset). b Representative image of two ORP1L positive lysosome-mitochondria contacts, indicated by white arrows, marked by the ER. Line-scan analysis of relative fluorescence intensities from the yellow line in merge is shown. Scale bar: 1 µm. c Representative time lapse image of ORP1L positive lysosomes forming stable contact with mitochondria indicated by white arrows on the first frame. Scale bar: 1 µm. d , e Percentage of Lamp1-mCherry and mCherry-ORP1L positive lysosomes in contact with mitochondria ( d ) and e minimum duration of these contacts in cells co-expressing mito-GFP. Cells from three independent experiments. Two-sided unpaired t -test. f , g Percentage of Lamp1-mCherry positive lysosomes in contact with mitochondria (mito-BFP) ( f ) and g minimum duration of these contacts in cells expressing GFP, GFP-ORP1L, or the D478A mutant. Cells from two independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. h – j Percentage of lysosome-mitochondria contacts that were marked by the ER: h when ORP1 expression was downregulated. Cells from three independent experiments, one-way ANOVA with Dunnett’s Multiple Comparison Test; i in ORP1L KO cells and control cells. Cells from three independent experiments, two-sided unpaired t -test; and j when VAPA and VAPB expression levels were downregulated. Data from three independent experiments, Two-sided unpaired t -test. k Cytosolic GAI-ΔANKORP1L can be recruited to GID1-Rab7 lysosomes upon GA 3 -AM treatment. l Representative image of a cell expressing mTq2-GAI-ΔANKORP1L and iRFP-GID1-Rab7 before and after GA 3 -AM treatment (10 µM). Scale bar: 10 µm. m Normalized fluorescence intensity of mTq2-GAI-ΔANKORP1L WT and D478A colocalizing with iRFP-GID1-Rab7 lysosomes at the indicated times before and after GA 3 -AM treatment. Cells from four independent experiments. n Relative ER fluorescence intensity at lysosome-mitochondria contacts at the indicated times before and after GA 3 -AM treatment (shaded area represent the area within one SEM). Cells from four independent experiments. Two-way ANOVA, Sidak’s multiple comparisons test. d – j , m , n All graphs show the mean ± SEM.

Article Snippet: The following constructs were made for this study: 3HA-Rab7, 3HA-Rab7, 3HA-Rab7 Q67L and 3HA-Rab7 T22N were generated by subcloning Rab7, Rab7 Q67L and Rab7 T22N into pcDNA3.1-3xHA-C1; mApple-Drp1 was made by replacing GFP by mApple (from mApple-TOM20) in the GFP-Drp1 plasmid; CFP-GAI-Sac1 was generated by subcloning Sac1 from GFP-ORPSAC1 into CFP-GAI(1-92) (gift from Takanari Inoue, Addgene # 37307) and CFP-GAI-Sac1 C392S was generated from CFP-GAI-Sac1 via site directed mutagenesis; GFP-ORPOSBP was made by replacing amino acid 334 to 950 of ORP1L from GFP-ORP1L by amino acid 184 to 809 of OSBP from mCherry-OSBP (gift from John Brumell, Hospital for Sick Children, Toronto); GFP-ORPOSBP HH/AA was made from GFP-ORPOSBP using site directed mutagenesis; iRFP-GID1-MFF was made by replacing Rab7 from iRFP-GID1-Rab7 by MFF from GFP-MFF (gift from Glia Voeltz, Addgene #49153); GFP-Sac1-MFF and GFP-Sac1 C392S-MFF were made by introducing wild-type and C392S mutant Sac1, from CFP-GAI-Sac1 and CFP-GAI-Sac1 C392S respectively, between GFP and MFF in the GFP-MFF plasmid.

Techniques: Expressing, Microscopy, Fluorescence, Mutagenesis, Comparison, Control

Journal: Nature Communications

Article Title: ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

doi: 10.1038/s41467-021-25621-4

Figure Lengend Snippet: a , b Normalized rate of mitochondrial division (number of mitochondrial divisions normalized by time and volume) in HeLa cells ( a ) expressing wild-type or mutants 3HA-Rab7. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test; b when VAPs expression levels were downregulated using siRNAs. Cells from three independent experiments. Two-sided unpaired t -test. The graphs show the mean ± SEM. c Live-cell imaging of HeLa cells expressing GFP-ORP1L and mApple-TOM20. The white and yellow arrows show an ORP1L positive lysosome-mitochondria contact and a mitochondrial fission event. Scale bar: 1 µm. d Percentage of mitochondrial division events that were marked by GFP-ORP1L (cells from two independent experiments) in HeLa cells. e Representative maximum projection images of mitochondrial morphology in HeLa cells treated with indicated siRNAs. Scale bars: 10 µm and 5 µm (inset). f – h Mitochondrial morphology was quantified for f mean area per mitochondrion, g mitochondrial number per region of interest (ROI), and h number of junctions per mitochondria. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. The graphs show the mean ± SEM. i , j Normalized rate of mitochondrial division of i HeLa cells treated with indicated siRNAs. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test and of j ORP1L WT and KO HeLa cells. Cells from three independent experiments. Two-sided unpaired t -test. The graphs show the mean ± SEM. k ORP1L mutants used and the loss of function caused by the mutations or deletions. l Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing GFP or GFP-ORP1L constructs and mApple-TOM20. The graphs show the mean ± SEM, cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. m Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing mTq2-GAI-ΔANKORP1L, iRFP-GID1-Rab7, and mApple-TOM20 before and after GA 3 -AM (10 µM) treatment. Cells were imaged from 5 to 20 min after GA 3 -AM treatment. Cells from three independent experiments. Two-sided unpaired t -test. The graphs show the mean ± SEM.

Article Snippet: The following constructs were made for this study: 3HA-Rab7, 3HA-Rab7, 3HA-Rab7 Q67L and 3HA-Rab7 T22N were generated by subcloning Rab7, Rab7 Q67L and Rab7 T22N into pcDNA3.1-3xHA-C1; mApple-Drp1 was made by replacing GFP by mApple (from mApple-TOM20) in the GFP-Drp1 plasmid; CFP-GAI-Sac1 was generated by subcloning Sac1 from GFP-ORPSAC1 into CFP-GAI(1-92) (gift from Takanari Inoue, Addgene # 37307) and CFP-GAI-Sac1 C392S was generated from CFP-GAI-Sac1 via site directed mutagenesis; GFP-ORPOSBP was made by replacing amino acid 334 to 950 of ORP1L from GFP-ORP1L by amino acid 184 to 809 of OSBP from mCherry-OSBP (gift from John Brumell, Hospital for Sick Children, Toronto); GFP-ORPOSBP HH/AA was made from GFP-ORPOSBP using site directed mutagenesis; iRFP-GID1-MFF was made by replacing Rab7 from iRFP-GID1-Rab7 by MFF from GFP-MFF (gift from Glia Voeltz, Addgene #49153); GFP-Sac1-MFF and GFP-Sac1 C392S-MFF were made by introducing wild-type and C392S mutant Sac1, from CFP-GAI-Sac1 and CFP-GAI-Sac1 C392S respectively, between GFP and MFF in the GFP-MFF plasmid.

Techniques: Expressing, Comparison, Live Cell Imaging, Construct

Journal: Nature Communications

Article Title: ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

doi: 10.1038/s41467-021-25621-4

Figure Lengend Snippet: a Representative image of a mitochondrial division marked by a GFP-2xP4M positive lysosome (yellow arrow) in HeLa cells expressing the indicated markers. PI4KA inhibitor GSK-A1 treatment allowed to better visualize PI(4)P at lysosomes. Scale bar: 1 µm. b Percentage of mitochondrial division events marked by lysosomes (cells from two independent experiments) and of lysosomes marked by GFP-2xP4M at mitochondrial division events. c In ORPSAC1, the ORD domain of ORP1L was replaced by the catalytic domain of Sac1 allowing the dephosphorylation of lysosomal PI(4)P. d Representative maximum projection images of mitochondrial morphology in HeLa cells overexpressing the indicated constructs. Scale bars: 10 µm and 5 µm (inset). e – g Mitochondrial morphology was quantified for e mean area per mitochondrion, f mitochondrial number per region of interest (ROI), and g number of junctions per mitochondria. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. h Normalized rate of mitochondrial division in HeLa cells overexpressing the indicated constructs. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. i Cytosolic GAI-Sac1 can be recruited to GID1-Rab7 lysosomes upon GA 3 -AM treatment to specifically deplete lysosomal PI(4)P. j , k Representative images of HeLa cells expressing CFP-GAI-Sac1 or the catalytic dead C392S mutant, iRFP-GID1-Rab7, and mCherry-2xP4M after GA 3 -AM treatment ( j ). Scale bars: 10 µm and 1 µm (inset). k Quantification of the lysosomal levels, normalized by the plasmalemmal levels, of 2xP4M. Cells from three independent experiments. Two-sided unpaired t -test. l Normalized rate of mitochondrial division before or after GA 3 -AM treatment in HeLa cells overexpressing iRFP-GID1-Rab7 and the indicated constructs. Cells from three independent experiments. Two-way ANOVA, Sidak’s multiple comparisons test. m Representative image of a HeLa cell expressing GFP-PI4K2B and Lamp1-mCherry with zoomed insert of white box in merge panel. Scale bar: 10 µm. n Normalized rate of mitochondrial division in HeLa cells treated with the indicated siRNAs. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test. e – h , k , l , n All graphs show the mean ± SEM.

Article Snippet: The following constructs were made for this study: 3HA-Rab7, 3HA-Rab7, 3HA-Rab7 Q67L and 3HA-Rab7 T22N were generated by subcloning Rab7, Rab7 Q67L and Rab7 T22N into pcDNA3.1-3xHA-C1; mApple-Drp1 was made by replacing GFP by mApple (from mApple-TOM20) in the GFP-Drp1 plasmid; CFP-GAI-Sac1 was generated by subcloning Sac1 from GFP-ORPSAC1 into CFP-GAI(1-92) (gift from Takanari Inoue, Addgene # 37307) and CFP-GAI-Sac1 C392S was generated from CFP-GAI-Sac1 via site directed mutagenesis; GFP-ORPOSBP was made by replacing amino acid 334 to 950 of ORP1L from GFP-ORP1L by amino acid 184 to 809 of OSBP from mCherry-OSBP (gift from John Brumell, Hospital for Sick Children, Toronto); GFP-ORPOSBP HH/AA was made from GFP-ORPOSBP using site directed mutagenesis; iRFP-GID1-MFF was made by replacing Rab7 from iRFP-GID1-Rab7 by MFF from GFP-MFF (gift from Glia Voeltz, Addgene #49153); GFP-Sac1-MFF and GFP-Sac1 C392S-MFF were made by introducing wild-type and C392S mutant Sac1, from CFP-GAI-Sac1 and CFP-GAI-Sac1 C392S respectively, between GFP and MFF in the GFP-MFF plasmid.

Techniques: Expressing, De-Phosphorylation Assay, Construct, Comparison, Mutagenesis

Journal: Nature Communications

Article Title: ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

doi: 10.1038/s41467-021-25621-4

Figure Lengend Snippet: a ORPOSBP is a chimeric protein made of the amino acids 1 to 335 of ORP1L fused to amino acids 184 to 809 of OSBP from Oryctalogus cuniculus . Domains are not to scale. b Representative image of a Hela cell expressing GFP-ORPOSBP and mRFP-Rab7. Zoomed images of dashed box shown on right. Scale bars: 10 µm and 5 µm (inset). c Time-lapse images of HeLa cells expressing GFP-ORPOSBP and mApple-TOM20. The yellow arrow shows a mitochondrial fission event marked by GFP-ORPOSBP. Scale bar: 1 µm. d , e Representative images of HeLa cells expressing the wild-type GFP-ORPOSBP or the lipid binding deficient HH/AA mutant with the PI(4)P probe mCherry-2xP4M ( d ). Scale bars: 10 µm and 1 µm (inset). e Quantification of the lysosomal levels of 2xP4M normalized by the plasmalemmal levels of the probe. The graphs show the mean ± SEM, cells from three independent experiments. Two-sided unpaired t -test. f Representative maximum projection images of mitochondrial morphology in HeLa cells treated with siRNA downregulating ORP1L and overexpressing GFP, GFP-ORPOSBP or the HH/AA ORPOSBP mutant and mApple-TOM20. Note that ORPOSBP wild-type expression, but not of the HH/AA mutant rescue the mitochondrial elongation and hyperfusion of the mitochondrial network caused by ORP1L depletion. Scale bars: 10 µm and 5 µm (inset). g – i Mitochondrial morphology was quantified for g mean area per mitochondrion, h mitochondrial number per region of interest (ROI), and i number of junctions per mitochondria. Cells from three independent experiments. One-way ANOVA with Dunnett’s Multiple Comparison Test, ns non statistically significant: g p = 0.6437, h p = 0.7205, and i p = 0.3046. The graphs show the mean ± SEM. j – l Mitochondrial morphology of Hela cells expressing GFP or GFP-ORPOSBP was quantified for j mean area per mitochondrion, k mitochondrial number per region of interest (ROI), and l number of junctions per mitochondria. Cells from three independent experiments. Two-sided unpaired t -test, ns non statistically significant. j p = 0.8011, k p = 0.6381 and l p = 0.9266. The graphs show the mean ± SEM.

Article Snippet: The following constructs were made for this study: 3HA-Rab7, 3HA-Rab7, 3HA-Rab7 Q67L and 3HA-Rab7 T22N were generated by subcloning Rab7, Rab7 Q67L and Rab7 T22N into pcDNA3.1-3xHA-C1; mApple-Drp1 was made by replacing GFP by mApple (from mApple-TOM20) in the GFP-Drp1 plasmid; CFP-GAI-Sac1 was generated by subcloning Sac1 from GFP-ORPSAC1 into CFP-GAI(1-92) (gift from Takanari Inoue, Addgene # 37307) and CFP-GAI-Sac1 C392S was generated from CFP-GAI-Sac1 via site directed mutagenesis; GFP-ORPOSBP was made by replacing amino acid 334 to 950 of ORP1L from GFP-ORP1L by amino acid 184 to 809 of OSBP from mCherry-OSBP (gift from John Brumell, Hospital for Sick Children, Toronto); GFP-ORPOSBP HH/AA was made from GFP-ORPOSBP using site directed mutagenesis; iRFP-GID1-MFF was made by replacing Rab7 from iRFP-GID1-Rab7 by MFF from GFP-MFF (gift from Glia Voeltz, Addgene #49153); GFP-Sac1-MFF and GFP-Sac1 C392S-MFF were made by introducing wild-type and C392S mutant Sac1, from CFP-GAI-Sac1 and CFP-GAI-Sac1 C392S respectively, between GFP and MFF in the GFP-MFF plasmid.

Techniques: Expressing, Binding Assay, Mutagenesis, Comparison

Journal: Nature Communications

Article Title: ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

doi: 10.1038/s41467-021-25621-4

Figure Lengend Snippet: a Soluble GAI-Sac1 can be recruited to mitochondrial fission site using GID1-MFF upon GA 3 -AM treatment, leading to dephosphorylation of PI(4)P at the mitochondrial division site. b Representative image of a HeLa cell expressing CFP-GAI-Sac1, iRFP-GID1-MFF (not imaged) and mApple-TOM20 before and after GA 3 -AM treatment (10 µM). Scale bar: 10 µm. c Normalized rate of mitochondrial division before and after GA 3 -AM (10 µM) treatment in HeLa cells overexpressing the wild-type CFP-GAI-Sac1 or the inactive C392S mutant, iRFP-GID1-MFF and mApple-TOM20. When treated with GA 3 -AM (10 µM) cells were imaged between 5 and 25 min of treatment. Cells from three independent experiments. Two-way ANOVA, Sidak’s multiple comparisons test. d In GFP-Sac1-MFF, Sac1 was fused to MFF to anchor it directly to the outer mitochondrial membrane leading to dephosphorylation of PI(4)P at the mitochondrial division site. e Representative maximum projection images of HeLa cells expressing GFP, GFP-MFF, GFP-Sac1-MFF or the catalytic inactive GFP-Sac1 C292S-MFF and mApple-TOM20. Inset shows the morphology of the mitochondrial network. Scale bars: 10 µm and 5 µm (inset). f – h Mitochondrial morphology was quantified for f mean area per mitochondrion, g mitochondrial number per region of interest (ROI), and h number of junctions per mitochondria. Cells from three independent experiments. One-way ANOVA with Tukey’s Multiple Comparison Test. i Representative images of ORP1L KO HeLa cells expressing the indicated constructs before and after GA 3 -AM (10 µM) treatment. Scale bar: 10 µm. j Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing the cytosolic mTq 2 -GAI-ΔANKORP1L D478A, mApple-TOM20, GFP-ORP1LΔORD, and iRFP-GID1-Rab7 before and after GA 3 -AM (10 µM) treatment. Cells from three independent experiments. Two-sided unpaired t-test. k Normalized rate of mitochondrial division in ORP1L KO HeLa cells expressing the cytosolic mTq 2 -GAI-ΔANKORP1L D478A, mApple-TOM20 and iRFP-GID1-Rab7 before and after GA 3 -AM (10 µM) treatment. Cells from three independent experiments. Two-sided unpaired t -test. c , f – h , j , k All graphs show the mean ± SEM. ns non-significant: f p = 0.7632, g p = 0.3592, h p = 0.7935, and k p = 0.5192.

Article Snippet: The following constructs were made for this study: 3HA-Rab7, 3HA-Rab7, 3HA-Rab7 Q67L and 3HA-Rab7 T22N were generated by subcloning Rab7, Rab7 Q67L and Rab7 T22N into pcDNA3.1-3xHA-C1; mApple-Drp1 was made by replacing GFP by mApple (from mApple-TOM20) in the GFP-Drp1 plasmid; CFP-GAI-Sac1 was generated by subcloning Sac1 from GFP-ORPSAC1 into CFP-GAI(1-92) (gift from Takanari Inoue, Addgene # 37307) and CFP-GAI-Sac1 C392S was generated from CFP-GAI-Sac1 via site directed mutagenesis; GFP-ORPOSBP was made by replacing amino acid 334 to 950 of ORP1L from GFP-ORP1L by amino acid 184 to 809 of OSBP from mCherry-OSBP (gift from John Brumell, Hospital for Sick Children, Toronto); GFP-ORPOSBP HH/AA was made from GFP-ORPOSBP using site directed mutagenesis; iRFP-GID1-MFF was made by replacing Rab7 from iRFP-GID1-Rab7 by MFF from GFP-MFF (gift from Glia Voeltz, Addgene #49153); GFP-Sac1-MFF and GFP-Sac1 C392S-MFF were made by introducing wild-type and C392S mutant Sac1, from CFP-GAI-Sac1 and CFP-GAI-Sac1 C392S respectively, between GFP and MFF in the GFP-MFF plasmid.

Techniques: De-Phosphorylation Assay, Expressing, Mutagenesis, Membrane, Comparison, Construct

Journal: Nature Communications

Article Title: ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

doi: 10.1038/s41467-021-25621-4

Figure Lengend Snippet: Mitochondrial division initiates at contacts with the ER, where the ER drives the constriction of mitochondrial membranes. This implicates an actin machinery that involves the ER protein INF2 and the mitochondrial Spire1C. The constriction allows the recruitment of Drp1 by adapters such as MFF and its oligomerization that further constrict mitochondrial membranes (non-represented in the cartoon). Lysosomes are then recruited to the division site in a process mediated by the Rab7-ORP1L-VAPs interaction that establishs contact sites with the ER ( 1 ). This allows the formation of a three-way contact between the ER, the lysosome and mitochondria at the division site that brings the lysosome in contact with mitochondria. Lysosome-mitochondria tethers are still unknown. At the Lysosome-mitochondria contact ( 2 ), we propose that ORP1L mediates the transfer of PI(4)P from lysosome to the division site. This model is supported by the impaired mitochondrial division when this transfer is inhibited or when PI(4)P is depleted at the mitochondrial division site.

Article Snippet: The following constructs were made for this study: 3HA-Rab7, 3HA-Rab7, 3HA-Rab7 Q67L and 3HA-Rab7 T22N were generated by subcloning Rab7, Rab7 Q67L and Rab7 T22N into pcDNA3.1-3xHA-C1; mApple-Drp1 was made by replacing GFP by mApple (from mApple-TOM20) in the GFP-Drp1 plasmid; CFP-GAI-Sac1 was generated by subcloning Sac1 from GFP-ORPSAC1 into CFP-GAI(1-92) (gift from Takanari Inoue, Addgene # 37307) and CFP-GAI-Sac1 C392S was generated from CFP-GAI-Sac1 via site directed mutagenesis; GFP-ORPOSBP was made by replacing amino acid 334 to 950 of ORP1L from GFP-ORP1L by amino acid 184 to 809 of OSBP from mCherry-OSBP (gift from John Brumell, Hospital for Sick Children, Toronto); GFP-ORPOSBP HH/AA was made from GFP-ORPOSBP using site directed mutagenesis; iRFP-GID1-MFF was made by replacing Rab7 from iRFP-GID1-Rab7 by MFF from GFP-MFF (gift from Glia Voeltz, Addgene #49153); GFP-Sac1-MFF and GFP-Sac1 C392S-MFF were made by introducing wild-type and C392S mutant Sac1, from CFP-GAI-Sac1 and CFP-GAI-Sac1 C392S respectively, between GFP and MFF in the GFP-MFF plasmid.

Techniques:

Journal: Nature Communications

Article Title: TGFβ signalling acts as a molecular brake of myoblast fusion

doi: 10.1038/s41467-020-20290-1

Figure Lengend Snippet: a , b , d , e DML-derived myocytes electroporated with MLC promoter driving expression of dominant negative (DN) variants of RAB11 ( b ) and RAB7 ( e ), together with membrane GFP (green) and nuclear mCherry (red). c , f , Column graph for a , b and d , e showing the population of electroporated myocytes containing the indicated number of nuclei relative to their controls (in %). g – i Functional rescue experiment where DML-derived myocytes were co-electroporated with a RFP-tagged form of TGFBR2 and an inducible (Tet-on) HA-tagged form of a constitutively active RAB7. g immunostaining against RFP showing the punctated expression of TGFBR2. h immunostaining against HA showing the diffuse expression of CA RAB7 (after Doxycyclin treatment). i Native fluorescence of H2B-BFP fusion protein, showing the nuclei within electroporated myocytes. j Merge of Fig. 5g–i. k Column graph showing the population of electroporated myocytes containing the indicated number of nuclei relative to their controls (in %) in each of the indicated conditions. Statistical analyses: DN RAB11: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 2.57; n = 14; Ctrl: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 2.07; n = 15; P -value <0.0001; DN RAB7: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 1.96; n = 19; Ctrl: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 2.46; n = 35; P -value < 0.0001; TGFBR2: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 1.63; n = 15; Ctrl: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 2.05; n = 23; P -value <0.0001; TGFBR2 + CA RAB7: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 1.84; n = 27; P -value=0.0019. *** P < 0.001. ** P < 0.01. Error bars in c , f , k : SEM. Scale bars: 50 μm. Source data are provided (see ‘Data availability’).

Article Snippet: A constitutively active form of human RAB7 (Q67L) was obtained from plasmid EGFP-Rab7A Q67L (a gift from Qing Zhong, Addgene plasmid # 28049 ).

Techniques: Derivative Assay, Expressing, Dominant Negative Mutation, Membrane, Functional Assay, Immunostaining, Fluorescence