The capacity of MOSPD2 to form ER–LD contact sites is necessary but not sufficient to regulate LDs. (A) Schematic representation of mScarlet-MOSPD2 constructs either WT (mScarlet-MOSPD2) or containing a deletion of the MSP domain (ΔMSP) or the CRAL-TRIO domain (ΔCRAL-TRIO), a mutation in the MSP domain (RD/LD) or in the CRAL-TRIO domain (W201E). For each construct, the LD tethering activity and the rescue (see panels below) are summarized as + or −. (B) Western Blot analysis of WT and MOSPD2 knock-out (KO#1) HeLa cells. MOSPD2 expression was rescued in MOSPD2 knock-out cells using mScarlet-MOSPD2 expression constructs either WT or mutant (mScarlet-MOSPD2 ΔCRAL-TRIO, W201E, and RD/LD). NT, non-transfected. (C) Representative confocal images of WT and MOSPD2 knock-out (KO#1) HeLa cells in which MOSPD2 expression was restored using mScarlet-MOSPD2 constructs (c, d, e, and f; green) depicted in panel A. As control, untransfected WT (a) and MOSPD2 knock-out (b) cells were imaged. LDs were stained with BODIPY 493/503 (magenta) and nuclei with Hoechst (blue). (D) Quantification of the number of LDs in cells shown in B. Data are displayed as Superplots showing the mean number of LDs per cell (small dots) and the mean number of LDs per independent experiment (large dots). Independent experiments (n = 5) are color-coded. Means and error bars (SD) are shown as black bars. Data were collected from 213 (WT), 200 (KO#1), 150 (KO + mScarlet-MOSPD2 WT), 238 (KO + mScarlet-MOSPD2 ΔCRAL-TRIO), 126 (KO + mScarlet-MOSPD2 W201E), and 118 (KO + mScarlet-MOSPD2 RD/LD) cells. One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; ***, P < 0.001; n = 5 independent experiments). (E) Schematic representation of mScarlet constructs containing the deletion of the CRAL-TRIO domain together with an insertion of the AH (AH-MOSPD2-ΔCRAL-TRIO), and of the GFP-AH-VAP-A chimera in which the AH of MOSPD2 was fused at the N-terminus of VAP-A. For both constructs, the LD tethering activity and the rescue (see panels below) are summarized as + or −. (F) Representative confocal images of WT and MOSPD2 knock-out (KO#1) of HeLa cells in which constructs (green) from panel E were expressed (c and d). As control, untransfected WT (a) and MOSPD2 knock-out (b) cells were imaged. LDs were stained with BODIPY 493/503 (magenta) and nuclei with Hoechst (blue). (G) Quantification of the number of LDs in cells shown in F. Data are displayed as Superplots showing the mean number of LDs per cell (small dots) and the mean number of LDs per independent experiment (large dots). Independent experiments (n = 5) are color-coded. Means and error bars (SD) are shown as black bars. Data were collected from 202 (WT), 192 (KO#1), 156 (KO + mScarlet-MOSPD2 WT), 147 (KO + mScarlet-AH-MOSPD2-ΔCRAL-TRIO), and 155 (KO + mScarlet-AH-VAP-A). One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; **, P < 0.01; ***, P < 0.001; n = 5 independent experiments). (C and F) Images were acquired on a spinning-disk confocal microscope (Nikon CSU-X1, 100× NA 1.4). Scale bars: 10 µm. The cell contour is shown with a white dotted line. Source data are available for this figure: SourceData F9.
Figure 9.

The capacity of MOSPD2 to form ER–LD contact sites is necessary but not sufficient to regulate LDs. (A) Schematic representation of mScarlet-MOSPD2 constructs either WT (mScarlet-MOSPD2) or containing a deletion of the MSP domain (ΔMSP) or the CRAL-TRIO domain (ΔCRAL-TRIO), a mutation in the MSP domain (RD/LD) or in the CRAL-TRIO domain (W201E). For each construct, the LD tethering activity and the rescue (see panels below) are summarized as + or −. (B) Western Blot analysis of WT and MOSPD2 knock-out (KO#1) HeLa cells. MOSPD2 expression was rescued in MOSPD2 knock-out cells using mScarlet-MOSPD2 expression constructs either WT or mutant (mScarlet-MOSPD2 ΔCRAL-TRIO, W201E, and RD/LD). NT, non-transfected. (C) Representative confocal images of WT and MOSPD2 knock-out (KO#1) HeLa cells in which MOSPD2 expression was restored using mScarlet-MOSPD2 constructs (c, d, e, and f; green) depicted in panel A. As control, untransfected WT (a) and MOSPD2 knock-out (b) cells were imaged. LDs were stained with BODIPY 493/503 (magenta) and nuclei with Hoechst (blue). (D) Quantification of the number of LDs in cells shown in B. Data are displayed as Superplots showing the mean number of LDs per cell (small dots) and the mean number of LDs per independent experiment (large dots). Independent experiments (n = 5) are color-coded. Means and error bars (SD) are shown as black bars. Data were collected from 213 (WT), 200 (KO#1), 150 (KO + mScarlet-MOSPD2 WT), 238 (KO + mScarlet-MOSPD2 ΔCRAL-TRIO), 126 (KO + mScarlet-MOSPD2 W201E), and 118 (KO + mScarlet-MOSPD2 RD/LD) cells. One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; ***, P < 0.001; n = 5 independent experiments). (E) Schematic representation of mScarlet constructs containing the deletion of the CRAL-TRIO domain together with an insertion of the AH (AH-MOSPD2-ΔCRAL-TRIO), and of the GFP-AH-VAP-A chimera in which the AH of MOSPD2 was fused at the N-terminus of VAP-A. For both constructs, the LD tethering activity and the rescue (see panels below) are summarized as + or −. (F) Representative confocal images of WT and MOSPD2 knock-out (KO#1) of HeLa cells in which constructs (green) from panel E were expressed (c and d). As control, untransfected WT (a) and MOSPD2 knock-out (b) cells were imaged. LDs were stained with BODIPY 493/503 (magenta) and nuclei with Hoechst (blue). (G) Quantification of the number of LDs in cells shown in F. Data are displayed as Superplots showing the mean number of LDs per cell (small dots) and the mean number of LDs per independent experiment (large dots). Independent experiments (n = 5) are color-coded. Means and error bars (SD) are shown as black bars. Data were collected from 202 (WT), 192 (KO#1), 156 (KO + mScarlet-MOSPD2 WT), 147 (KO + mScarlet-AH-MOSPD2-ΔCRAL-TRIO), and 155 (KO + mScarlet-AH-VAP-A). One-way ANOVA with Tukey’s multiple comparisons test (ns, not significant; **, P < 0.01; ***, P < 0.001; n = 5 independent experiments). (C and F) Images were acquired on a spinning-disk confocal microscope (Nikon CSU-X1, 100× NA 1.4). Scale bars: 10 µm. The cell contour is shown with a white dotted line. Source data are available for this figure: SourceData F9.

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