Acute Manipulation of Diacylglycerol Reveals Roles in Nuclear Envelope Assembly & Endoplasmic Reticulum Morphology
Figure 5
1,2- and 1,3-DAG rescue the fragmented NE phenotype.
(A) Confocal images of live HeLa cells 1 min after addition of small unilamellar vesicles (SUVs) containing BODIPY-PtdCho and unsaturated 1,2 DAG (80∶20 mole% respectively). Incorporation of SUVs (green) into interphase and metaphase (white arrows) cells are shown. (B) LBR localisation during mitosis in rapalogue-treated, LBR and DGKε-expressing HeLa cells after addition at metaphase of SUVs containing PtdCho and unsaturated 1,2 DAG (80∶20 mole%). NE reformation (yellow arrows) was rescued. (C) Ultrastructure of the NE (yellow arrow) of the same cell at cytokinesis imaged using CLEM (Fig. S4). LBR localisation in green, DGKε in red. Serial sections are shown in Movie S8. (D) Comparison of 3D models reconstructed from serial images of DAG-depleted (left panel) and DAG-rescued (right panel) cells shows the NE reformed in the presence of 1,2 DAG. (E–G) Similar results as in (A–C) respectively were obtained with SUVs of the non-C1 domain-binding DAG isomer 1,3 DAG. (H) CLEM images of a rapalogue-treated, dark LBR and DGKε-expressing HeLa cell fixed at cytokinesis, after addition of (60∶40 mole %) SUVs with BODIPY-PtdCho and unsaturated 1,3 DAG. Incorporation of the SUVs into cell membranes in green, DGKε in red. EM images show 1,3 DAG completely rescued NE reformation. Images representative of n = 11 experiments. Scale bars: confocal 10 μm, CLEM as indicated on the images.