Optogenetic Miro cleavage reveals direct consequences of real-time loss of function inDrosophila

Abstract

Miro GTPases control mitochondrial morphology, calcium homeostasis and regulate mitochondrial distribution by mediating their attachment to the kinesin and dynein motor complex. It is not clear, however, how Miro proteins spatially and temporally integrate their function as acute disruption of protein function has not been performed. To address this issue, we have developed an optogenetic loss of function 'Split-Miro' allele for precise control of Miro-dependent mitochondrial functions in Drosophila. Rapid optogenetic cleavage of Split-Miro leads to a striking rearrangement of the mitochondrial network, which is mediated by mitochondrial interaction with the microtubules. Unexpectedly, this treatment did not impact the ability of mitochondria to buffer calcium. While Split-Miro overexpression is sufficient to augment mitochondrial motility, sustained photocleavage shows Split-Miro is surprisingly dispensable to maintain elevated mitochondrial processivity. Furthermore, functional replacement of endogenous Miro with Split-Miro identifies its essential role in the regulation of locomotor activity in adult flies, demonstrating the feasibility of tuning animal behaviour by real-time loss of protein function.

Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons

“…Temperature and CO2 were maintained at 37°C and 5%, respectively, via a temperature unit with CO2 control and a microscope enclosure (Okolab). Axonal transport was recorded approximately 100 m away from the cell body and kymographs were produced by first straightening 100 m of axonal tract (approximately in mid axon) using the Straighten plugin and followed by the Velocity Measurement Tool in ImageJ, as described previously (Annuario et al, 2022;Mattedi et al, 2023). Mitochondrial speed, run length and number of total mitochondria were analysed directly from the kymographs.…”

“…Response curves were aligned at the base of the response peak and the fluorescence intensity at every time point was normalised to the value of the first frame. Fluorescence 'peak' was defined as the maximum foldchange value reached during imaging and the time to reach the peak was calculated as the t between the 'peak' timepoint and the time of first response to stimulation, as reported previously (Mattedi et al, 2023). Time to decay was defined as the time necessary for the fluorescence signal to decline by 80% of the peak value.…”