Fig. 6: Diverse MDSs connect with peroxisomes in neurons.

a Confocal image, representative of (d), of a neuronal cell expressing the rsEGFP2 fused to the OMM (rs2-OMM, magenta) and immunostained for the peroxisomal protein PEX14 (green). The inset shows the dendrites, immunostained for MAP2 (gray). b Two-color STED and confocal comparison of multiple mitochondria and peroxisomes, representative of (d). STED image, in contrast to the confocal comparison, allows to appreciate the outer mitochondrial membrane and the peroxisomal membrane. c Three representative examples of MDSs and line profiles measured across #1, #2, and #3 in the images. (Top) MDS from the tip of a mitochondrion, with OMM and PEX14 signals colocalizing in the vesicle membrane, indicating the possible biogenesis of a new pre-peroxisome; (middle) MDS where OMM is localized inside PEX14; (bottom) MDS with OMM and PEX14 signals, indicating another example of possible mitochondria-peroxisome exchange mechanisms. d Distribution of MDS area divided into PEX⁺ (green) and PEX⁻ (gray). Data were collected from six DIV7–9 hippocampal neurons from two independent experiments. MDS_OXPHOS− = 142; MDS_OXPHOS+ = 233. Kolmogorov–Smirnov test, **P < 0.01. e Two-color STED image of rs2-OMM (magenta) and the peroxisomal protein PEX14 (immunostaining, green), superimposed to a confocal image of the OXPHOS complex (immunostaining, yellow). The image is a representative example of the data plotted in (g). f Three examples of MDSs and line profiles at the indicated positions from (e), showing the absence of OXPHOS in PEX⁺ structures. g Box plot of the normalized frequency of MDS types with respect to the presence of PEX14 and OXPHOS (OMM⁺PEX14⁺OXPHOS⁺: mean = 0.51, SD = 0.15, N_tot = 71; OMM⁺PEX14⁺OXPHOS⁻: mean = 0.2, SD = 0.15, N_tot = 31; OMM⁺PEX14⁻OXPHOS⁻: mean = 0.18, SD = 0.12, N_tot = 29; OMM⁺PEX14⁻OXPHOS⁺: mean = 0.11, SD = 0.09, N_tot = 16). Data were collected from four neurons, N_tot = 147 MDSs. For the box plots in (d, g), the center line represents the median, the box spans the interquartile range (IQR; 25th to 75th percentiles), and whiskers extend to 1.5× IQR.