Fig. 1: MDS heterogeneity in neurons.
a Confocal image of the neuronal mitochondrial network, stained through the C-terminal localization peptide of the mitochondrial outer membrane protein OMP25 (OMM) fused to the Halo-tag (Halo-OMM, red hot) and the dendritic filaments, immunostained for the microtubule-associated protein 2 (MAP2, blue). The inset shows mitochondrial area distribution as measured in STED (yellow) or confocal (gray); the dashed line shows the threshold value (Ath = 0.086 µm2), chosen to discriminate between mitochondria and mitochondrial-derived structures (MDSs). Data were collected from 31 DIV7–9 hippocampal neurons from more than three independent experiments. NSTED = 1391 organelles, NConfocal = 136 organelles. b STED (top) and confocal (middle) images of mitochondria (A > Ath) and MDSs (A < Ath), as representative examples of the data plotted in (c, d). Normalized intensity line profiles from indicated lines (bottom), as measured in confocal (gray line) and STED (black dots), and Gaussian fits of STED data (black line). c MDS area distribution as a histogram with bin width 0.01 µm2. Data were collected from 21 DIV7–9 hippocampal neurons from more than three independent experiments. NMDVs = 637, median = 0.025 µm2. d MDS aspect ratio (AR) distribution as a histogram with bin width 0.1 µm2. Data were collected from 21 DIV7–9 hippocampal neurons from four independent cultures. The dotted line shows the threshold value Ath discriminating between sticks (AR < Ath) and vesicles (AR > Ath). NMDVs = 637. e STED images (left) and normalized line profiles (right) of representative vesicular and tubular structures, with FWHM of 89 nm and 102 nm, respectively (dots are data points; lines are Gaussian fits). The images are representative examples of the data plotted in (f). f Number density distribution of MDSs across different neuronal compartments. Data are expressed as mean ± standard deviation. No statistically significant differences have been observed. Data were collected from 25 FOVs taken from four cells from two independent experiments. Student’s t-tests: Pall neurites = 0.68; Paxons = 0.85; Pdendrites = 0.30.
