pet22b(+) vector  (Thermo Fisher)

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Schematic representation of STARD3. The MENTAL domain in blue presents four transmembrane helices (dark blue). The sequence of the phospho-FFAT motif is highlighted in a red box. The four serine residues phosphorylated within and around the FFAT motif are numbered. The consensus sequence for the GSK3 recognition site is also shown. B-D: HA-tagged GSK3P (green) was expressed alone (B) or together with WT (C) and S209A mutant (D) STARD3 (magenta) in MCF7 cells. GSK3P and STARD3 were labeled with anti-HA and anti-STARD3 antibodies, respectively. The subpanels are higher magnification images of the area outlined in white. The overlay panel shows merged images of green, magenta and blue (nuclei labelled with Hoechst-33258) images. Scale bars: 1O µm. Inset scale bars: 2 µm. E: Pearson’s correlation coefficients between GSK3P-HA and STARD3 (WT or S209A) staining. Data are displayed as Superplots with Pearson’s correlation coefficient for individual cells (small dots) and the mean per independent experiment (large dots). Number of cells: GSK3P-HA-STARD3: 29; GSK3P-HA-STARD3 S209A: 33, from three independent experiments. Independent experiments are color-coded. Mean values with error bars (SD) are shown. Unpaired t-test (**P < 0.01). F, H: Western blot analysis of HCC1954 (G) and MCF7/STARD3 (H) cells treated or not with the GSK3 inhibitor CHIR99021 (5 µM; overnight). STARD3 protein level (Total) and S209 phosphorylation (pS209) were analyzed. Right: quantification of relative S209 phosphorylation levels. Means± SD. Student’s t-test (***, P < 0.001; n = 3 independent experiments). G: Western blot analysis of control MCF7 cells (left) and MCF7 cells stably overexpressing STARD3 (right). 1-J: Western blot analysis of HCC1954 (I) and MCF7/STARD3 (J) cells transfected with control siRNAs (siCtrl) or siRNAs targeting GSK3a (siGSK3a), GSK3p (siGSK3p), or both (siGSK3a + siGSK3a). Bottom: quantification of relative S209 phosphorylation levels. Means± SD. One-way ANOVA with Dunnett’s multiple comparison test(*, P < 0.05; **, P < 0.01; ***, P < 0,001, n = 3 independent experiments).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Sequencing, Mutagenesis, Labeling, Staining, Western Blot, Phospho-proteomics, Control, Stable Transfection, Transfection, Comparison

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Western blot analysis of MCF? cells overexpressing TIP60 treated or not with the GSK3 inhibitor CHIR99021 (5 µM; overnight). TIP60 protein levels (Total) and S586 phosphorylation (pS586) were analyzed. B: Western blot analysis of Hela/STARD3 cells treated overnight with different concentration of CHIR99021 (1, 2 and 5 µM) or left untreated. STARD3 protein level (Total) and S209 phosphorylation (pS209) were analyzed. C: (a) Western blot analysis of HCC1954 cells treated with 5 µM of CHIR99021 for 2, 4, 6, 8 and 16 h, or left untreated. STARD3 protein levels (Total) and S209 phosphorylation (pS209) were analyzed. (b) Quantification of relative S209 phosphorylation levels. Means± SD. One-way ANOVA with Dunnett’s multiple comparison test(**, P < 0.01; n = 3 independent experiments). (c) Representative images of HCC1954 cells expressing WT STARD3 and treated with the GSK3 inhibitor CHIR99021 for 2, 4, 6, 8 and 16 h, or left untreated. Cells were labelled with an anti-LAMP1 antibody (magenta), an anti-STARD3 antibody (green) and Hoescht-33258 for nuclei (blue).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Western Blot, Phospho-proteomics, Concentration Assay, Comparison, Expressing

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Schematic representation of the phospho-FFAT sequence of STARD3, highlighting the different phosphorylated serines. B-C: Western blot analysis of MCF7 cells expressing WT and mutant (S209A; S213A; S217A; S221A; S213A + S217A + S221A) STARD3. C: quantification of relative S209 phosphorylation levels. Means ± SD. One-way ANOVA with Dunnett’s multiple comparison test(***, P < 0.001; n = 3 independent experiments). D: Schematic representation of the recombinant proteins used in the in vitro kinase assay. E: Coomassie blue staining of recombinant GSK313, cSTD3 WT, cSTD3 pS209, cSTD3 pS213 and cSTD3 S213E after SOS-PAGE. F: Western blot analysis of in vitro kinase assay for cSTD3 WT, cSTD3 pS209, cSTD3 pS213, and cSTD3 S213E incubated with recombinant GSK313 (right) or without it (left). Right: quantification of the relative S209 phosphorylation levels. Means ± SD. One-way ANOVA with Dunnett’s multiple comparison test(***, P < 0.001; ****, P < 0.0001; n = 3 independent experiments).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Sequencing, Western Blot, Expressing, Mutagenesis, Phospho-proteomics, Comparison, Recombinant, In Vitro, Kinase Assay, Staining, Incubation

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A-C: lmmunoprecipitation (GFP-Trap) experiments between GFP-VAP-A (A), GFP-VAP-B (B) or GFP-MOSPD2 (C) and Flag-tagged STARD3. Approximatively 5 µg of total protein extract was analyzed by Western blot using anti-STARD3, anti-pS209-STARD3, anti-GFP and anti-GAPDH antibodies. lmmunoprecipitated proteins were analyzed using anti-STARD3, anti-pS209-STARD3 and anti-GFP antibo­ dies. D: lmmunoprecipitation experiment between endogenous STARD3 and MOSPD2 in HCC1954 cells. Cells were treated or not with CHIR99021, and proteins extracted. lmmunoprecipitation was performed using control lgG or anti-STARD3 antibodies. Total protein extract and immunoprecipitated proteins were analyzed by Western blot using anti-STARD3, anti-pS209-STARD3, anti-MOSPD2, and anti-GAPDH antibodies. E-I: Hela cells expressing GFP-VAP-A (E-G, I) and GFP-VAP-A KO/MD (H) (green) were either untransfected (A) or transfected with STARD3 WT (F, H, I) or STARD3 S209A (G). Cells were left untreated (E, F-H) or treated with CHIR99021 (I). STARD3 was labeled using anti-STARD3 antibodies (F-I, magenta), and nuclei stained with Hoechst (blue). Insets show higher magnification images of the areas outlined in white. Scale bars: 10 µm. Inset scale bars: 2 µm. The overlay panels show merged green, magenta and blue images. J: Pearson’s correlation coefficients between VAP-A (WT or KO/MD mutant) and STARD3 (WT or STARD3S209A) in cells treated or not with CHIR99021. Data are displayed as Superplots with Pearson’s correlation coefficient for individual cells (small dots) and the mean per independent experiment (large dots). Number of cells: VAP-A-STARD3: 28; VAP-A-STARD3 treated with CHIR99021: 28; VAP-A-STARD3S209A: 30, VAP-A KD/MD-STARD3: 27, from three independent experiments. Independent experiments are color-co­ ded. Means± SD. ANOVA with Tukey’s multiple comparison test(***, P < 0.001).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Western Blot, Control, Immunoprecipitation, Expressing, Transfection, Labeling, Staining, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A, G: HeLa cells expressing GFP-VAP-A (A, B; green) or GFP-VAP-B (C-G) were either untransfected (A-D) or transfected with STARD3 WT (E, G) or STARD3 S209A (F). Cells were left untreated (C, E, F) or treated with CHIR99021 (B, D, G). STARD3 was labeled using anti-STARD3 antibodies (magen­ ta), and nuclei were stained with Hoechst (blue). Insets show higher magnification images of the areas outlined in white. Scale bars: 10 µm. Inset scale bars: 2 µm. The overlay panels show merged green, magenta and blue images. In A-D, endogenous STARD3 levels were too low to be detected with anti-STARD3 antibodies. H: Pearson’s correlation coefficients between VAP-B and STARD3 (WT or S209A) in cells treated or not with CHIR99021. Each dot represents a single cell (number of cells: VAP-B-STARD3: 26; VAP-B-STARD3 treated with CHIR99021: 30; VAP-B-STARD3 S209A: 33, from three independent experi­ ments). Means and error bars (SD) are shown. ANOVA with Tukey’s multiple comparison test(**, P < 0.01).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Expressing, Transfection, Labeling, Staining, Comparison

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A-C: HCC1954 cells either non-transfected (NT) (A) or transfected with control siRNAs (siCtrl) (B), or siRNAs targeting STARD3 (siSTARD3) (C) and treated or not with CHIR99021 (5 µM, overnight). STARD3 (green) and LAMP1 (magenta) were labeled with antibodies, and nuclei (blue) stained with Hoechst. The subpanels are higher magnification images of the area outlined in white. Scale bars: 10 µm. Inset scale bars: 2 µm. D: Clustering index of LAMP1-positive vesicles of samples shown in A-C. Data are displayed as Superplots showing the clustering index per cell (small dots) and its mean per independent experiment (large dots). Number of cells: HCC1954-WT-NT: 41, HCC1954-siCtrl-NT: 47, HCC1954-siS­ TARD3-NT: 45, HCC1954-WT-CHIR99021: 48, HCC1954-siCtrl-CHIR9902: 39, HCC1954-siSTARD3-CHIR9902: 51; from three independent experiments. Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. One-way ANOVA with Tukey’s multiple comparison test(*, P < 0.05; **, P < 0.01; ****, P < 0.0001; n = 3 independent experiments). Scale bars: 10 µm. Inset scale bars: 2 µm. E, F: MCF? cells expressing STARD3 (C) and STARD3 S209A (D) were left untreated (NT; left) or treated with CHIR99021 (right). Cells were labelled with anti-STARD3 antibodies (total STARD3; green) and phospho-specific antibodies (pS209 STARD3; magenta). Nuclei were stained with Hoechst (blue). Subpanels show higher magnification images of the area outlined in white. Scale bars: 10 µm. Inset scale bars: 2 µm. G: Clustering index of STARD3-positive vesicles of samples shown in E, F. Data are displayed as Superplots showing the clustering index per cell (small dots) and its mean per independent experiment (large dots). Number of cells: MCF7-STARD3-NT: 41, MCF7-STARD3-CHIR99021: 52, MCF7-STARD3 S209A-NT: 33, MCF7-STARD3 S209A-CHIR99021: 32, from four independent experiments. Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. One-way ANOVA with Tukey’s multiple comparison test(****, P < 0.0001; n = 4 independent experiments). Scale bars: 10 µm. Inset scale bars: 2 µm.

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Transfection, Control, Labeling, Staining, Comparison, Expressing

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Cells were labelled with anti-STARD3 (green), anti-LAMP1 antibodies (magenta) with nuclei stained with Hoechst (blue). Images were acquired with a confocal Spinning Disk microscope with a Live-SR Super Resolution module and analyzed with CellProfiler. Scale bar: 10 µm. B: Image segmentation: LE/Lys were segmented based on STARD3 staining (or LAMP1 staining in ) using StarDist, trained on a custom dataset. Cell contours were manually segmented. C: Clustering Index quantification: using the segmentation data of cells and LE/Lys, the proportion of LE/Lys in contact with at least one other LE/Lys per cell was determined (Cellprofiler, Module: MeasureOjectNeighbors, module method: Adjacent). The clustering index ranges from O (all LE/Lys are isolated) to 1 (all LE/Lys are in contact with at least one other).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Staining, Microscopy, Isolation

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: WT MCF7 cells (A), MCF7 cells expressing STARD3 WT (B-F) or FFAT-motif deficient mutants (STARD3 FYAA (F), STARD3 b.FFAT (G)) were left untreated (left) or treated with CHIR99021 (5 µM, overnight; right). Cells were labelled with anti-STARD3 antibodies (green) (A-G) and in magenta with: anti-LAMP1 antibodies (A-B) to label LE/Lys, anti-EEA1 antibodies (C) to label early endosomes, anti-GM130 antibodies (D) to label the Golgi apparatus, anti-calnexin antibodies (E) to label the ER or phospho-specific antibodies (pS209 STARD3, F-G). Nuclei were stained with Hoechst (blue). Subpanels show higher magnification images of the area outlined in white. Scale bars: 10 µm. Inset scale bars: 2 µm.

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A-C : Hela cells (A), U2OS cells (B) and COS-7 cells (C) expressing STARD3 WT were left untreated (left) or treated with CHIR99021 (5 µM, overnight) (right). Cells were labelled with anti-STARD3 antibodies (green) and anti-LAMP1 antibody (magenta). Nuclei were stained with Hoechst (blue). Subpanels show higher magnification images of the area outlined in white. Images were acquired with a SP8-UV confocal microscope. Scale bars: 10 µm. Inset scale bars: 2 µm. D, E: Representative images of MCF7 cells expressing STARD3 WT, STARD3 S209A, STARD3 S213A, STARD3 S217A, STARD3 S221A, or STARD3 S213A-S217A-S221A. Cells were labelled with anti-STARD3 antibodies (green) and Hoechst (blue). Scale bars: 10 µM. E: Quantification of LE/Lys cluste­ ring in cells shown in D. Data are displayed as Superplots showing the clustering index per cell (small dots) and its mean per independent experiment (large dots). Number of cells: MCF7-STARD3: 70, MCF7-STARD3 S209A: 31, MCF7-STARD3 S213A: 31, MCF7-STARD3 S217A: 18, MCF7-STARD3 S221A: 32, MCF7-STARD3 S213A-S217A-S221A: 31, from seven independent experiments). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. One-way ANOVA with Tukey’s multiple comparison test(**, P < 0.01; ***, P < 0.001; n = 7 independent experiments). F-J: MCF7 (A) expressing STARD3 WT (F), STARD3 S209A (G), STARD3 t.FFAT (H), STARD3 S209At.START (I), or STARD3 SD/PA S213A (J) were left untreated (left) or treated with CHIR99021 (5 µM, overnight; right). Cells were labelled with anti-STARD3 antibodies (green). Nuclei were stained with Hoechst (blue). Scale bars: 10 µm. Inset scale bars: 2 µm.

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Expressing, Staining, Microscopy, Comparison

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Western blot analysis of MCF7/STARD3 cells transfected with control siRNAs (siCtrl), siRNAs targeting VAP-A (siVAP-A), VAP-8 (siVAP-8), MOSPD2 (siMOSPD2), and the three together. B: MCF7/STARD3 cells either non-transfected (a) or transfected with control siRNAs (b), or siRNAs targeting VAP-A (c), VAP-B (d), MOSPD2 (e) or the three together (f). STARD3 (green) was labeled using anti-STARD3 antibodies, and nuclei (blue) stained with Hoechst. The subpanels are higher magnification images of the area outlined in white. Scale bars: 10 µm. Inset scale bars: 2 µm. C: Clustering index of STARD3-positive vesicles of samples shown in B. Data are displayed as Superplots showing the clustering index per cell (small dots) and its mean per independent experiment (large dots). Number of cells: MCF7/STARD3-NT: 35, MCF7/STARD3-siCtrl: 33, MCF7/STARD3-si­ VAP-A: 35, MCF7/STARD3-siVAP-B: 25, MCF7/STARD3-siMOSPD2: 36, MCF7/STARD3-siVAP-A/B/MOSPD2: 39, from three independent experi­ ments). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. One-way ANOVAwith Tukey’s multiple compa­ rison test(****, P < 0.0001; n = 3 independent experiments). D: Super-resolution imaging (SIM2) of an MCF7 cell expressing STARD3 and treated with CHIR99021. The subpanel on the right shows a higher magnification (2x) image of the area outlined in white. Scale bar: 5 µm. E-G: 3D rendering of LE/Lys (magenta), ER (green), mitochondria (brown) in control Hela cells (E), cells expressing WT STARD3 (F), and cells expres­ sing the STARD3 FA/YA mutant (G), imaged by FIB-SEM.

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Western Blot, Transfection, Control, Labeling, Staining, Imaging, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Schematic representation of the different STARD3 mutants used. B-E: Representative images of MCF7 cells expressing WT STARD3 (B), STARD3 S209A (C), STARD3 SD/PA (D), STARD3 t.START (E) left untreated (left) or treated with CHIR99021 (right), and labelled with an anti-STARD3 antibody (green) and with Hoechst (nuclei; blue). Scale bars: 10 µm. F: Clustering index of STARD3-positive vesicles of samples shown in B-E and . Data are displayed as Superplots showing the clustering index per cell (small dots) and its mean per independent experiment (large dots). Number of cells: STARD3-NT: 37, STARD3-CHIR99021: 37, STARD3 S209A-NT: 25, STARD3 S209A-CHIR99021: 32, STARD3 t.FFAT-NT: 40, STARD3 ts FFAT-CHIR99021: 29, STARD3 SD/PA-NT: 35, STARD3 SD/PA-CHIR99021: 28, STARD3 t.START-NT: 43, STARD3 t.START-CHIR99021: 44, from five independent experiments. Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. One-way ANOVA with Tukey’s multiple comparison test(****, P < 0.0001; n = 5 independent experiments). G: Schematic representation of STARD3NL mutants used in the study. The MENTAL domain in light blue contains 4 transmembrane helices (dark blue) and a Phospho-FFAT motif (red). The chimeric construct STARD3NL-START domain is composed of STARD3NL fused to the START domain of STARD3. H-J: Representative images of MCF7 cells expressing WT STARD3NL (H), STARD3NL t.FFAT (I), and the STARD3NL-START chimeric construct (J) left untreated (left) or treated with CHIR99021 (right), and labelled with an anti-STARD3NL antibody (green) and with Hoescht-33258 (nuclei; blue). Scale bars: 10 µm. K: Clustering index of STARD3NL-positive vesicles of samples shown in H-J. Data are displayed as Superplots showing the clustering index per cell (small dots) and its mean per independent experiment (large dots). Number of cells: STARD3NL-NT: 78, STARD3NL-CHIR99021: 66, STARD3NL t.FFAT-NT: 32, STARD3NL t.FFAT-CHIR99021: 32, STARD3NL-START-NT: 33, STARD3NL-START-CHIR99021: 37, from four independent experiments). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. One-way ANOVA with Tukey’s multiple comparison test(*, P < 0.05; ****, P < 0.0001; n = 4 independent experiments). L: Schematic representation of chimeric constructs: one consisting of the TMEM192 transmembrane fragment fused to the START domain of STARD3, and another combining the amino-terminal region of LAMTOR1 with the START domain of STARD3 (referred to as Lyso-START). M-N: Representative images of MCF7 cells expressing TMEM192-START chimera (M), and Lyso-START (N), left untreated (left) or treated with CHIR99021 (right), and labelled with an anti-STARD3 antibody (green) and with Hoescht-33258 (nuclei; blue). Scale bars: 10 µm. O: Clustering index of STARD3-positive vesicles of samples shown in M-N. Data are displayed as Superplots showing the clustering index for individual cell (small dots) and the mean per independent experiment (large dots). Number of cells: STARD3-NT: 29, STARD3-CHIR99021: 24, TMEM192-START chimera-NT: 27, Lyso-START-NT: 30 from three independent experiments. Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. One-way ANOVA with Dunett’s multiple comparison test(****, P < 0.0001; n = 3 independent experiments).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Expressing, Comparison, Construct

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: TEM images (a) of control Hela cells (A), Hela cells expressing STARD3 (B), and Hela cells expressing STARD3 FA/YA (C). Scale bars: 500 nm. Schematic representation (b) of images shown in (a) indicate the ER, endosomes, and intraluminal membranes in dark, light, and medium gray, respec­ tively. Mt: mitochondria; Ne: nucleus.

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: MCF7 cells expressing STARD3 WT left untreated (left) or treated with CHIR99021 (5 µM, overnight; right) (A) and MCF7 cells expressing STARD3NL-START (B), TMEM192-START (C), and Lyso-START (D) were labelled with anti-STARD3 antibodies (green) and with anti-LAMP1 antibodies to label LE/Lys (magenta). Nuclei were stained with Hoechst (blue). Subpanels show higher magnification images of the area outlined in white. Scale bars: 10 µm. Inset scale bars: 2 µm.

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Schematic representation of the different recombinant proteins used. B: Coomassie blue staining of the recombinant START domain of STARD3 (WT and KD/KD mutant) and the MSP domain of VAP-B proteins after SDS-PAGE. C: Sedimentation velocity (SV) profile obtained by analytical ultracentrifugation (SV-AUC). The sample contained either 15 µM (blue) or 37.5 µM (purple) of the recombinant protein. The recombinant START domain of STARD3 has a theoretical sedimentation coefficient (Stheo) of 2.41 Sand displayed an average sedimentation coefficient (S20,w) of 2.35 S, consistent with a stable monomeric form at both concentrations. D: Ribbon diagram of the START domain of STARD3. The positions of the two mutated residues, K260 and K281, are highlighted. Key structural features, including omega loop 1 (01) and three beta strands (P1, p2, P3), are also indicated (PDB ID: 1EM2) . E: Far-UV CD spectrum of purified cSTD3 and cSTD3 KD/KD constructs in 20 mM Tris pH 7.4, 120 mM NaF buffer at room temperature.

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Recombinant, Staining, Mutagenesis, SDS Page, Sedimentation, Analytical Ultracentrifugation, Purification, Construct

Journal: bioRxiv

Article Title: STARD3 coordinates Endoplasmic Reticulum-late endosome/lysosome contacts and organelle positioning through a GSK3-regulated phosphorylation switch

doi: 10.1101/2025.05.29.656564

Figure Lengend Snippet: A: Schematic representation of the different STARD3 mutants used. B-E: Representative images of MCF7 cells expressing WT STARD3 (J), STARD3 KD/KD (K), STARD3 S209A (L), and STARD3 S209A KD/KD (M). In J and K, cells were left untreated (left) or treated with CHIR99021 (right). Cells were labelled with an anti-STARD3 antibody (green) and with Hoechst (nuclei; blue). Scale bars: 10 µm. F: Clustering index of STARD3-positive vesicles of samples shown in B-E. Data are displayed as Superplots showing the clustering index per cell (small dots) and its mean per independent experiment (large dots). Number of cells: STARD3 WT-NT: 65, STARD3 WT-CHIR99021: 59, STARD3 KD/KD-NT: 37, STARD3 KD/KD-CHIR99021: 32, STARD3 S209A: 57, STARD3 S209A KD/KD: 47, from six independent experiments). Independent experiments are color-coded. Means and error bars (SD) are shown as black bars. One-way ANOVA with Tukey’s multiple comparison test (*, P < 0.05; ••••, P < 0.0001; n = 6 independent experiments).

Article Snippet: Hurley , was opened with NdeI and NcoI, the following primers 5’-TTT AAG AAG GAG ATA TAC AT ATG GGC CAT CAT CAT CAT CAT CAC ATG GGG TCT GAC AAT GAA TC -3’; 5’ GAT TCA TTG TCA GAC CCC ATG TGA TGA TGA TGA TGA TGG CCC ATA TGT ATA TCT CCT TCT TAAA -3’ were hybridized and cloned by the SLiCE method to generate the pET22b-START-STARD3 V2 (216-445) vector. pES002 MBP-GSK3β S9A-HA-His was a gift from Jesse Zalatan (Addgene plasmid # 196184; http://n2t.net/addgene:196184 ; RRID:Addgene_196184) ( ). pcDNA3 HA-GSK3β was a gift from Jim Woodgett (Addgene plasmid # 14753; http://n2t.net/addgene:14753 ; RRID:Addgene_14753) ( ).

Techniques: Expressing, Comparison