Image acquisition and analysis

Cells growing on “Y”‐shaped micropatterned substrates were selected on the basis of shape (visualised with phalloidin or a reporter fill) and nuclei (visualised with DAPI) to ensure that only single cells properly attached to the micropatterns were used. The mitochondrial channel (visualised with MitoTracker) was not used to select cells before the acquisition. The mitochondrial area was thresholded, and Sholl analysis of mitochondrial distribution was performed using a custom‐made ImageJ plugin (Lopez‐Domenech et al, 2016). Mitochondrial signal was quantified within shells radiating out from the soma at 1‐μm intervals and the cumulative distribution of mitochondrial signal or Mitochondrial Probability Map (MPM) was plotted per genotype. The distance at which 50, 90 and 95% of the total mitochondrial mass is found (Mito⁵⁰, Mito⁹⁰ and Mito⁹⁵ values, respectively) was calculated per each cell by interpolation. One average, Mito⁹⁵ value was calculated per genotype per experiment and used to quantify a final Mito⁹⁵ value (n = experiments) in figures. In case of overexpression experiments, total number of cells was used as the n (n = cells).