MOSPD2 is dynamically recruited to ER–LD contact sites. (A) CLEM of HeLa/GFP-MOSPD2 cells. a: fluorescence microscopy image; b: EM image; c: correlation of GFP-MOSPD2 and EM images (scale bar: 2 µm); d: composite showing higher magnification images of the area outlined in black in c (scale bar: 500 nm); bottom right: interpretation scheme showing contacts between organelles; ER and lipid droplets are in cyan and pink, respectively. Mitochondria and endosomes/lysosomes are in light yellow and gray, respectively. (B–D) FRAP analysis of MOSPD2 and PLIN2 mobility. GFP-MOSPD2 (B) and GFP-PLIN2 (C) expressing cells were treated with OA and labeled with LipidTOX. GFP fluorescence was photobleached in the area outlined in white, and images acquired every second over a 1-min period. Scale bars: 2 µm. (D) Lineplot showing the relative fluorescence intensity in the photobleached region of GFP-MOSPD2 (green) and GFP-PLIN2 (purple) expressing cells. The grey curve shows the relative fluorescence intensity of GFP-positive LDs that were not bleached. Means and error bars (SD) of relative fluorescence intensities of 56 (GFP-MOSPD2), 57 (GFP-PLIN2), and 72 (unbleached control) regions of interest from 20, 13, and 26 cells, respectively. Data from three independent experiments. (E and F) HeLa cells expressing GFP-MOSPD2 (green) were treated with OA and labeled with anti-PLIN3 antibodies (magenta). Images were acquired by confocal microscopy (Leica SP8, 63× NA 1.4; E), or by STED super-resolution microscopy (F). MOSPD2 and PLIN3 were heterogeneously distributed around LDs. Scale bar: 10 µm (insets, 2 µm) in E and 5 µm (insets, 1 µm) in F. Subpanels on the right are higher magnification images of the area outlined. The overlay panel shows merged channels. In E, linescan shows fluorescence intensities of the green and magenta channels along the white circular arrow of the overlay subpanel (i.e., at the surface of LDs). (G) HeLa cells expressing GFP-MOSPD2 were imaged live during LDs induction (stained with LipidTOX) by OA addition. The white arrow shows an enrichment of MOSPD2 signal before the appearance of LipidTOX staining. The yellow arrow shows the growth of a LD positive for MOSPD2 before the start of the induction. Images were acquired every 90 s (t0-900) on a spinning-disk confocal microscope (Nikon CSU-X1, 100× NA 1.4). Scale bar: 2 µm.
Figure 3.

MOSPD2 is dynamically recruited to ER–LD contact sites. (A) CLEM of HeLa/GFP-MOSPD2 cells. a: fluorescence microscopy image; b: EM image; c: correlation of GFP-MOSPD2 and EM images (scale bar: 2 µm); d: composite showing higher magnification images of the area outlined in black in c (scale bar: 500 nm); bottom right: interpretation scheme showing contacts between organelles; ER and lipid droplets are in cyan and pink, respectively. Mitochondria and endosomes/lysosomes are in light yellow and gray, respectively. (B–D) FRAP analysis of MOSPD2 and PLIN2 mobility. GFP-MOSPD2 (B) and GFP-PLIN2 (C) expressing cells were treated with OA and labeled with LipidTOX. GFP fluorescence was photobleached in the area outlined in white, and images acquired every second over a 1-min period. Scale bars: 2 µm. (D) Lineplot showing the relative fluorescence intensity in the photobleached region of GFP-MOSPD2 (green) and GFP-PLIN2 (purple) expressing cells. The grey curve shows the relative fluorescence intensity of GFP-positive LDs that were not bleached. Means and error bars (SD) of relative fluorescence intensities of 56 (GFP-MOSPD2), 57 (GFP-PLIN2), and 72 (unbleached control) regions of interest from 20, 13, and 26 cells, respectively. Data from three independent experiments. (E and F) HeLa cells expressing GFP-MOSPD2 (green) were treated with OA and labeled with anti-PLIN3 antibodies (magenta). Images were acquired by confocal microscopy (Leica SP8, 63× NA 1.4; E), or by STED super-resolution microscopy (F). MOSPD2 and PLIN3 were heterogeneously distributed around LDs. Scale bar: 10 µm (insets, 2 µm) in E and 5 µm (insets, 1 µm) in F. Subpanels on the right are higher magnification images of the area outlined. The overlay panel shows merged channels. In E, linescan shows fluorescence intensities of the green and magenta channels along the white circular arrow of the overlay subpanel (i.e., at the surface of LDs). (G) HeLa cells expressing GFP-MOSPD2 were imaged live during LDs induction (stained with LipidTOX) by OA addition. The white arrow shows an enrichment of MOSPD2 signal before the appearance of LipidTOX staining. The yellow arrow shows the growth of a LD positive for MOSPD2 before the start of the induction. Images were acquired every 90 s (t0-900) on a spinning-disk confocal microscope (Nikon CSU-X1, 100× NA 1.4). Scale bar: 2 µm.

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