Journal: The EMBO Journal

Article Title: STARD3 regulates lysosome positioning and contacts via a GSK3-controlled phosphorylation switch

doi: 10.1038/s44318-026-00705-3

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 labeled 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 – H ) MCF7 ( A ) expressing STARD3 WT ( D ), STARD3 S 209 A ( E ), STARD3 ΔFFAT ( F ), STARD3 SD/PA S 213 A ( G ) or STARD3 S 209 A ΔSTART ( H ) were left untreated (left) or treated with CHIR99021 (5 µM, overnight; right). Cells were labeled with anti-STARD3 antibodies (green). Nuclei were stained with Hoechst (blue). Scale bars: 10 µm. Inset scale bars: 2 µm. ( I , J ) Representative images of MCF7 cells expressing STARD3 WT, STARD3 S 209 A, STARD3 S 213 A, STARD3 S 217 A, STARD3 S 221 A, or STARD3 S 213 A-S 217 A-S 221 A. Cells were labeled with anti-STARD3 antibodies (green) and Hoechst (blue). Scale bars: 10 µM. ( E ) Quantification of LE/Lys clustering 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 S 209 A: 31, MCF7-STARD3 S 213 A: 31, MCF7-STARD3 S 217 A: 18, MCF7-STARD3 S 221 A: 32, MCF7-STARD3 S 213 A-S 217 A-S 221 A: 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 = 3–7 independent experiments; WT vs S 209 A, P = 6.8 × 10 −3 ; WT vs S 213 A, P = 3 × 10 −3 ; WT vs S 217 A, P = 0.99; WT vs S 221 A, P = 0.99; WT vs S 213 A-S 217 A-S 221 A, P = 10 −3 ; S 209 A vs S 213 A, P = 0.99; S 209 A vs S 213 A-S 217 A-S 221 A, P = 0.95; S 213 A vs S 213 A-S 217 A-S 221 A, P = 0.99). .

Article Snippet: Imaging was performed with a transmission electron microscope (Philips CM12) coupled to an Orius 1000 CCD camera (Gatan).

Techniques: Expressing, Labeling, Staining, Microscopy, Comparison

Journal: The EMBO Journal

Article Title: STARD3 regulates lysosome positioning and contacts via a GSK3-controlled phosphorylation switch

doi: 10.1038/s44318-026-00705-3

Figure Lengend Snippet: ( A , B ) Flotation assay. Liposomes (750 µM total lipids) composed of DOPC, DOPC/MPB-PE (97:3), DOPC/DOPS (70:30), or DOPC/DOPS/MPB-PE (67:30:3) and doped with 0.2% NBD-PA were mixed with DTT-free C STD3 ( A ) or C STD3 KD/KD ( B ) (0.75 µM) for 1 h in HK buffer at 25 °C. After centrifugation, the liposomes were collected at the top of sucrose cushions and analyzed by SDS-PAGE. The amount of membrane-bound protein was determined using the content of lane 5 (100% total) as a reference based on the SYPRO Orange intensity. Data are represented as mean ± s.e.m. ( n = 4) with single data points. ( C ) Principle of liposome pull-down assays. Proteins were immobilized on magnetic NTA-Ni 2+ beads thanks to their 6His tag and incubated with fluorescent liposomes. After washing, the bound liposomes were imaged. ( D , E , G ) Representative confocal images of NTA-Ni 2+ beads either not bound to recombinant proteins (no Prot.) or bound to recombinant wild-type ( D , E , G ) or KD/KD mutant ( G ) START domain or MSP domain ( D , E ). Beads were incubated with fluorescent neutral liposomes ( D ) or fluorescent negatively charged liposomes ( E , G ) (green). Top: Confocal sections showing liposome fluorescence; bottom: merged fluorescence and brightfield images of the beads. Spinning-disk confocal microscope (Nikon CSU-X1, 100× NA 1.4) images. Scale bars: 10 µm. Quantification of liposome recruitment on NTA-Ni 2+ beads. NBD fluorescence was measured around the beads. Data are displayed as Superplots, showing the relative fluorescence intensity per bead (small dots) and the mean per independent experiment (large dots). Number of beads: START WT ( D ) 72, ( E ) 75, ( G ) 104; MSP: ( D ) 47, ( E ) 58; START KD/KD: ( G ) 104; no Prot.: ( D ) 52, ( E ) 78, ( G ) 114; from three independent experiments). Independent experiments are color-coded. Means and error bars (SD) are represented by black bars. One-way ANOVA with Tukey’s multiple comparison test (***, P < 0.001; ****, P < 0.0001; n = 3 independent experiments; ( D ): START WT vs MSP, P = 0.96; START WT vs No Prot., P = 0.71; MSP vs No Prot., P = 0.55; ( E ): START WT vs MSP, P = 5 × 10 −4 ; START WT vs No Prot., P = 4 × 10 −4 ; ( G ): START WT vs MSP, P = 10 −4 ; START WT vs No Prot., P < 10 −4 ). ( F ) DLS experiments. DTT-free C STD3 (0.5 µM) was mixed with liposomes (50 µM lipids) composed of DOPC, DOPC/MPB-PE (97:3), DOPC/DOPS (70:30), or DOPC/DOPS/MPB-PE (67:30:3) in HK buffer at 25 °C. The mean radius (dots) and polydispersity (shaded area) of the liposome suspension were measured for 2 h. Bottom panel: size distribution before (gray bars) and after the reaction (colored bars). The data are representative of 4 independent experiments. ( H ) DLS experiments. DTT-free C STD3 or C STD3 KD/KD (0.5 µM) was mixed with liposomes (50 µM lipids) composed of DOPC/DOPS/MPB-PE (67:30:3) in HK buffer at 25 °C. The mean radius (dots) and polydispersity (shaded area) of the liposome suspension were measured for 2 h. Bottom panel: size distribution before (gray bars) and after the reaction (colored bars). The data are representative of 4 independent experiments. .

Article Snippet: Imaging was performed with a transmission electron microscope (Philips CM12) coupled to an Orius 1000 CCD camera (Gatan).

Techniques: Liposomes, Centrifugation, SDS Page, Membrane, Incubation, Recombinant, Mutagenesis, Fluorescence, Microscopy, Comparison, Suspension