Fig. 5: SM partitions differently into early endosome subcompartments.
From: Visualizing suborganellar lipid distribution using correlative light and electron microscopy
a, CLEM data analysis: images were acquired by FM and EM at medium magnification. After correlation, the ROI was chosen based on the fluorescent signal of LDL (blue) and Tf (magenta). The lipid signal is shown in orange. At the ROI, a tomogram was acquired at high magnification. Tomograms were further analysed if the endosomes contained BM (orange arrow), ILVs (blue arrow) and a RT (magenta arrow). Based on the tomogram, 3D models were derived. The z projection of the 3D model outlines is shown. Membrane domains in the model are classified into BM (orange), ILVs (blue) and RTs (magenta) and the respective classes were blurred to create class-specific masks. Scale bars, 10 µm (overview images) and 500 nm (ROIs). b, Three representative endosomes are shown with the fluorescent overlay, classified 3D model and model overlays with the fluorescent signal (grey) of SM, Tf or LDL from left to right. Scale bars, 500 nm. Right: the relative densities of all fluorescent signals (SM, Tf and LDL) over all membrane classes: RT (magenta), ILVs (blue) and the BM (orange). c, A representative endosome of a cell treated with the bifunctional fatty acid and the corresponding quantification of the fluorescent signals. Scale bar, 500 nm. d, The relative fluorescent densities are plotted for all analysed endosomes for SM and the metabolically labelled samples (FA). The mean densities are shown for the different domains: RT (magenta), BM (orange) and ILV (blue) and for the respective fluorescent signals of lipid, Tf and LDL. Maximum possible fold enrichments are indicated by the dotted horizontal lines for the BM (orange), RT (magenta) and ILVs (blue). No enrichment is indicated by the grey line at 1.0. Error bars are shown as the 95% confidence interval of the mean values. Single data points are shown in grey. P values are indicated and were determined using a two-sided Student’s t-test. The exact P values are 0.00192, 0.82109, 0.14332, 0.12113, 0.53060, 0.48663, 0.10007, 0.64835 and 0.08744 from left to right in d. Two independent biological replicates were performed both for SM (n (endosomes) = 13) and the fatty acid (n (endosomes) = 12). **P < 0.01, *P < 0.05, non-significant (n.s.) P > 0.05.
