Fig. 4: Mitochondria occur more frequently at nascent dendritic branching sites in developing neurons and are aligned to a central position prior to branch induction.

a Mitochondrial occurrence (mitochondria per µm dendrite segment) in non-branching dendritic segments and in dendritic segments undergoing dendritic branch induction, as determined by quantitative analyses of 3D-time-lapse imaging (t = 0 s of branch induction; branching, 1 µm segments evaluated; frame rate, 1 full z-stack/10 s; all mitochondria included (i.e. irrespective of their speed)). (b, c) Merges of MIPs of t = 0 s frame (b) and t = +60 s frame (c) of 3D-time-lapse spinning disc microscopic recordings of neuronal morphology highlighted by plasma membrane visualization with farnesylated mCherry (transfection with mCherryF-reported scrambled RNAi plasmids at DIV4; outlined by dashed line at site of protrusion to enhance visibility) and mitochondrial position detected by MitoTracker (m; green circle) in DIV7 primary rat hippocampal neurons (see Supplementary Fig. 13 for images of individual fluorescence channels). Bars, 500 nm. d–f Quantitative analyses of mitochondrial positioning in temporal and spatial relation to newly developing dendritic branch sites in control DIV7 rat hippocampal neurons at three different time points, 60 s prior to branch induction (t = −60 s; d), during dendritic branch initiation (t = 0 s; e) and 60 s after the induction of a dendritic branch (t = +60 s; f; branch establishment and outgrowth). Dendritic segment length of the three zones (distal, lateral segment towards dendrite tip; central, directly at dendritic branching site; proximal, lateral segment towards cell body) 1 µm each. Color-coded visual presentation (upper panels) (for color coding see legend) and in form of quantitation graphs (lower panels). Data, mean ± SEM visualized as bar/dot plots. a n = 24 forming dendritic branching sites at 1 µm segments from 24 neurons evaluated at t = 0 s; n = 24 non-branching dendritic segments (cumulated length of non-branching segments, 138.47 µm) of the same neurons. d–f n = 48 independent dendritic branch sites from 24 neurons, respectively (each evaluated for the 3 time points). Statistical significances, Mann-Whitney (a) and Kruskal-Wallis/Dunn´s post-test (d–f). ** P < 0.01; **** P < 0.0001. For exact P value ≥ 0.0001 see figure.