Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study.

Article Snippet: Rabbit monoclonal anti‐Rab7 , Cell Signaling Technology , Cat#9367; RRID:AB_1904103.

Techniques: Recombinant, In Situ, CRISPR, Control, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Golgi-endosome transport mediated by M6PR facilitates release of antisense oligonucleotides from endosomes

doi: 10.1093/nar/gkz1171

Figure Lengend Snippet: GCC2 localizes to LEs in HeLa and SGVA cells treated with PS-ASO. ( A ) Immunofluorescent staining of GCC2 in HeLa cells. Scale bar, 10 μm. ( B ) Immunofluorescent staining of GCC2 in HeLa cells incubated with 2 μM Cy3-labeled PS-ASO 446654 for 18 h. Scale bars, 10 μm. The regions boxed with dashed lines are magnified in the insets. ( C ) Immunofluorescent staining of GCC2 and LAMP1 in HeLa cells. Scale bars, 10 μm. ( D ) Immunofluorescent staining of GCC2 and LAMP1 in HeLa cells incubated with Cy3-labeled PS-ASO 446654 for 18 h. Scale bars, 10 μm. ( E ) 3D image of GCC2, PS-ASO and LAMP1 co-localization in HeLa cells incubated with Cy3-labeled PS-ASO 446654 for 18 h. The image was compiled from 25 sections taken at 0.1 μm depth. Two vesicles are marked with red and white arrows. ( F ) Immunofluorescent staining of GCC2 in GFP-Rab7-expressing SVGA cells incubated with 2 μM Cy3-labeled PS-ASO 446654 for 18 h. Scale bars, 5 μm. The regions boxed with dashed lines are magnified in the insets. The co-localization of GCC2 with PS-ASOs inside an LE is marked with an arrow.

Article Snippet: For live cell imaging, GFP-Rab7-expressing SVGA cells grown in glass-bottom dishes were incubated with 2 μM PS-ASO 446654 for 6 h. For GFP-M6PR-CD expression, plasmid (RG201277, Origene) was transfected into HeLa cells for 24 h. Cells were reseeded into glass-bottom dish and incubated for ON.

Techniques: Staining, Incubation, Labeling, Expressing

Journal: Nucleic Acids Research

Article Title: Golgi-endosome transport mediated by M6PR facilitates release of antisense oligonucleotides from endosomes

doi: 10.1093/nar/gkz1171

Figure Lengend Snippet: M6PR co-localizes with PS-ASOs and GCC2 in LEs in SVGA and HeLa cells. ( A ) Immunofluorescent staining of M6PR-CI in GFP-Rab7-expressing SVGA cells. Scale bars, 10 μm. In the far right panel, the region boxed with a dashed line is magnified in the inset. Co-localization between Rab7 and M6PR-CI is marked by arrows. ( B ) Immunofluorescent staining of M6PR-CI in GFP-Rab7-expressing SVGA cells incubated with 2 μM Cy3-labeled PS-ASO 446654 for 6 h. Scale bars, 10 μm. The regions boxed with dashed lines are shown at higher magnification in insets, and co-localization is indicated by arrows. ( C ) Quantification of co-localization between Rab7 and M6PR-CI in cells incubated with or without ASO for 6 h. Error bars are standard deviations. P -value was calculated based on unpaired t -test. ****, P < 0.0001. ( D ) Immunofluorescent staining of M6PR-CI and GCC2 in HeLa cells incubated with 2 μM Cy3-labeled PS-ASO 446654 for 12 h. An example of co-localization in a cytoplasmic area is boxed. ( E ) 3D image of the boxed area in panel D. Two foci where M6PR-CI, GCC2, and PS-ASO co-localize are marked by arrows.

Article Snippet: For live cell imaging, GFP-Rab7-expressing SVGA cells grown in glass-bottom dishes were incubated with 2 μM PS-ASO 446654 for 6 h. For GFP-M6PR-CD expression, plasmid (RG201277, Origene) was transfected into HeLa cells for 24 h. Cells were reseeded into glass-bottom dish and incubated for ON.

Techniques: Staining, Expressing, Incubation, Labeling

Journal: Nucleic Acids Research

Article Title: Golgi-endosome transport mediated by M6PR facilitates release of antisense oligonucleotides from endosomes

doi: 10.1093/nar/gkz1171

Figure Lengend Snippet: M6PR-CI co-localizes with PS-ASOs inside LEs and on the membranes of LEs. ( A ) Immunofluorescent staining of M6PR-CI in GFP-Rab7-expressing SVGA cells incubated with 2 μM PS-ASO 446654 for 12 h. Scale bars, 10 μm. The regions boxed with dashed lines are shown at higher magnification in insets. M6PR-CI co-localization with a distinct PS-ASO-containing focus inside the LE is marked with an arrow. ( B ) Immunofluorescent staining of M6PR-CI in GFP-Rab7-expressing SVGA cells incubated with 2 μM PS-ASO 446654 for 12 h. Co-localization of M6PR and PS-ASOs as a distinct structure on the LE membrane is marked by arrows. Scale bars, 5 μm. ( C ) Live cell imaging of a SGVA cell incubated with PS-ASO 446654 for 6 h. The nuclear border is marked by a dashed line. The boxed PS-ASO-containing LE in the lower right is magnified in panel D. ( D ) Upper images: Snapshots taken during live cell imaging. Potential PS-ASO release events are marked by arrows. Lower plots: Signal intensity profiles for the LE across the lines as indicated in the upper panels. Arrows indicate ASOs outside the LE body.

Article Snippet: For live cell imaging, GFP-Rab7-expressing SVGA cells grown in glass-bottom dishes were incubated with 2 μM PS-ASO 446654 for 6 h. For GFP-M6PR-CD expression, plasmid (RG201277, Origene) was transfected into HeLa cells for 24 h. Cells were reseeded into glass-bottom dish and incubated for ON.

Techniques: Staining, Expressing, Incubation, Live Cell Imaging

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: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min ( N = 3; 16 to 28 cells per condition); scale bar, 10 μm. ( Ca ) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. ( E and F ) Affibody-chase experiments in (E) siControl Trastuzumab-Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quantitation represents cytoplasmic HER2 fluorescence intensity ( N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Representative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to α V β 6 IACs and coordinates HER2 and α V β 6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between α V β 6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. ( B ) Trastuzumab-Resistant Cells: GDI2 is excluded from α V β 6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/α V β 6 cross-talk is impaired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and α V β 6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Activity Assay, Membrane, Migration, Activation Assay, Clinical Proteomics

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Differential gene expression data (RNA-seq) for the GDI2 / RAB5A / RAB7A / ERBB2 / ITGB6 cluster in normal breast tissue ( n = 403; light gray) and breast invasive carcinoma ( n = 1097; dark gray). Data were extracted from the TNMplot database ( tnmplot.com ). Black lines in violin blots represent the median. Mann-Whitney test. ( B ) Volcano plot showing statistical analysis (ANOVA) of RNA-seq gene expression data of patients with HER2+ breast cancer from the METABRIC cohort expressing high (Right) and low (Left) levels of ITGB6 (Q1 versus Q4). Significant genes (dark gray); nonsignificant genes (light gray); relevant genes are highlighted in purple. ( C ) Visual representation of GO terms analysis (ClueGO, cellular compartment) of genes highly and significantly expressed in tumors expressing high levels of ITGB6 (Q4). Colors represent specific merged GO term groups, node size represents the level of significance of each GO term, and clustering and edge length represent functionally grouped networks based on kappa score. ( D ) OS of patients with HER2+ breast cancer and with high (above median) expression of ITGB6 , expressing high (red) or low (black) levels of GDI2 , ERBB2 , RAB5A , and RAB7A . ( E and F ) Differential ITGB6 gene expression (gene chip) in patients with HER2+ breast cancer subdivided according to therapeutic response to trastuzumab. (E) Initial pathological complete response (responder) versus residual disease after completing therapy (nonresponder) ( n = 77 patients). (F) RFS at 5 years (responder) versus samples relapsed before 5 years (nonresponder) ( n = 24 patients). Two-sided Student’s t test. [(A), (E), and (F)] Statistical significance: * P < 0.05; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Gene Expression, RNA Sequencing, MANN-WHITNEY, Expressing, Clinical Proteomics