Francesca Mattedi, Ethlyn Lloyd-Morris, Frank Hirth, Alessio Vagnoni
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.
Methods to observe loss of function (LoF) phenotypes are used to study many biological processes. Although important tools for elucidating gene function, disruption of genes by genomic mutations or RNA interference (RNAi) often does not have the spatiotemporal resolution to capture the direct cellular and organismal consequences of LoF and to report specifically on a protein’s primary function. The observed “end-point” phenotypes might thus be the result of compensatory mechanisms to loss of protein, and gene pleiotropy means that, even in cell culture models, it is often difficult to dissect the causality of the observed phenotypes.
Materials and method
Generation of plasmid DNA
The new constructs produced in this study are reported in S1 Table and were generated either through restriction enzymes mediated cloning or NEBuilder HiFi DNA Assembly (NEB) using the primers listed in S2 Table. The plasmid inserts were amplified by PCR using the Q5 Hot-Start High-Fidelity 2X Master Mix (NEB). Site-directed mutagenesis was performed using the Q5 Site-directed mutagenesis kit (NEB) following the manufacturer’s instructions. The fidelity of all assembled constructs was verified by Sanger sequencing (Eurofins Genomics).
Design of a photocleavable Miro variant in Drosophila
To gain real-time spatiotemporal control of Miro LoF, we created a Miro variant that contains the LOV2-Zdk1 protein pair (Fig 1A and 1B) that undergoes light-induced dissociation upon exposure to blue light [14–16] and so is predicted to achieve rapid and reversible Miro LoF through protein photocleavage (Fig 1B).
Using optogenetics to implement a real-time LoF paradigm by targeting Miro, we show that collapse of the mitochondrial network is an immediate response to Miro photocleavage in S2R+ cells, which temporally precedes the defects observed in mitochondrial trafficking. We found that Miro overexpression increases the proportion and the processivity of mitochondria transported in the processes of S2R+ cells. Surprisingly, although sustained Split-Miro photocleavage reverted the proportion of transported mitochondria to control levels, the velocities and run lengths of the motile organelles were largely unaffected. Interestingly, Split-Miro photocleavage decreases the number, but not the velocities and run length, of motile mitochondria in adult fly neurons in vivo, suggesting that a potentially similar mechanism might account for the regulation of mitochondrial motility in the processes of S2R+ cells and in adult neurons.
We thank Tito Calì, Manolis Fanto, Gohta Goshima, Marc-David Ruepp, Tom Schwarz, Zu-Hang Sheng, Konrad Zinsmaier, and Alex Whitworth for sharing reagents, the Fly Facility of the Department of Genetics, University of Cambridge for help with Drosophila embryo injections, the BFK Lab for assistance with the behavioural assays, the Wohl Cellular Imaging Centre at King’s College London for help with light microscopy, and the Bloomington Drosophila Stock Center for fly stocks. We thank members of the Vagnoni lab, Joe Bateman and Simon Bullock for critically reading the manuscript.
Citation: Mattedi F, Lloyd-Morris E, Hirth F, Vagnoni A (2023) Optogenetic cleavage of the Miro GTPase reveals the direct consequences of real-time loss of function in Drosophila. PLoS Biol 21(8): e3002273. https://doi.org/10.1371/journal.pbio.3002273
Academic Editor: Anna Akhmanova, Utrecht University, NETHERLANDS
Received: October 14, 2022; Accepted: July 22, 2023; Published: August 17, 2023
Copyright: © 2023 Mattedi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This work was supported by a NC3Rs David Sainsbury fellowship (NC/N001753/2) and NC3Rs SKT grant (NC/T001224/1), an Academy of Medical Sciences Springboard Award (SBF004/1088), an ARUK King’s College London Network Centre Grant (ARUK-NC2020-KCL), a van Geest Fellowship in Dementia and Neurodegeneration, and a van Geest Studentship to A.V. A MRC-DTP Studentship supports E.L.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: AR, aspect ratio; DART, Drosophila ARousal Tracking; fps, frame per seconds; HRP, horseradish peroxidase; LoF, loss of function; MERCS, mitochondria-ER contacts site; RNAi, RNA interference; ROI, region of interest; wt-Miro, wild-type Miro