RT Journal Article SR Electronic T1 Optogenetic Miro cleavage reveals direct consequences of real-time loss of function in Drosophila JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.10.01.510462 DO 10.1101/2022.10.01.510462 A1 Mattedi, Francesca A1 Lloyd-Morris, Ethlyn A1 Hirth, Frank A1 Vagnoni, Alessio YR 2022 UL http://biorxiv.org/content/early/2022/10/02/2022.10.01.510462.abstract AB 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.Competing Interest StatementThe authors have declared no competing interest.