Clonal Imaging of Mitochondria in the Dissected Fly Wing

Abstract

Mitochondria are essential for long-term neuronal function and survival. They are maintained in neurons, including long axonal stretches, through dynamic processes such as fission, fusion, biogenesis, and mitophagy. Here, we describe a protocol for the in-depth morphological analysis of individual mitochondria in axons in vivo. Most mitochondrial analysis of axons is currently performed in vitro with neurons in a developmental state. Therefore, an understanding of the axonal mitochondrial network during aging in fully differentiated neurons and the long-term consequence of gene knockout is often not developed. By using a clonal system paired with fluorescent genetically encoded markers in theDrosophilawing, we can visualize individual neurons (out of the whole bundle), including their long axons and the mitochondria that they contain, using confocal imaging. The clonal system also allows visualization of neurons with genetic perturbations that would otherwise be lethal if present in the whole organism, allowing investigators to bypass lethality. This protocol can further be adapted to measure the physiological and biochemical state of the mitochondria. Mitochondrial morphology and health in axons are tightly linked to aging, axon injury, and neurodegeneration; therefore, this method can be used to investigate mitochondrial dysfunction associated with novel genes or those linked to neurodegenerative disease and axonopathy.

COPI-regulated mitochondria-ER contact site formation maintains axonal integrity

“… 32 , 33 The chemical mutagen ethyl methanesulfonate was used to induce DNA mutations at random genomic locations, and the MARCM (mosaic analysis with a repressible cell marker) system was used to induce homozygous mutant clones in F 1 progeny so that neurons and mitochondria they contain could be visualized using genetically encoded markers at single-cell resolution. 32 , 34 , 35 Importantly this clonal approach allows for in-depth characterization of genes that would otherwise be lethal if knocked out in the whole animal or nervous system. The l ( 3 ) 2750 mutant was subsequently named αCOP − after whole-genome sequencing confirmed a 28 bp deletion and the introduction of a premature stop codon in the αCOP gene as the phenotype-causing lesion ( Figure S1 A).…”

“…With the exception of mitochondrial motility and Ca 2+ reporters, wings and legs were dissected with microdissection scissors, mounted in halocarbon oil between a microscope slide and coverslip and imaged immediately. 35 For imaging of mitochondrial motility and Ca 2+ reporters, Drosophila were live-mounted between a microscope slide and a tape-spaced coverslip to create a custom-sized chamber. 40 Wings were painted down with halocarbon oil and imaged immediately for 240 s at 1 frame per second.…”

Analysis of Mitochondrial Dynamics in AdultDrosophilaAxons

“…By substituting UAS-mito::GFP with other fluorescent marker elements, these protocols can be easily adapted to investigate other important internal structures such as lysosomes, autophagosomes, other vesicles, the endoplasmic reticulum, or the cytoskeleton (see Protocol: Clonal Imaging of Mitochondria in the Dissected Fly Wing [Maddison et al 2022]; Protocol: Live Imaging of Mitochondria in the Intact Fly Wing [Mattedi et al 2022]). Using the same progeny from genetic crosses indicated here, it is also possible to study synapse-residing mitochondria by dissecting and mounting whole Drosophila legs.…”

“…We introduce two methodological approaches for the in-depth study of mitochondrial morphology and dynamics in adult axons in vivo (see Protocol: Clonal Imaging of Mitochondria in the Dissected Fly Wing [Maddison et al 2022]; Protocol: Live Imaging of Mitochondria in the Intact Fly Wing [Mattedi et al 2022]). These are useful both for hypothesis-driven approaches Vagnoni and Bullock 2018) and for discovering new modulators of mitochondrial function through completely unbiased genetic screening (Smith et al 2019b;Lin et al 2021).…”

“…The stereotypical organization of sensory neurons in the wing facilitates excellent visualization of mitochondria throughout the whole neuron, with axons bundled together in the proximal region of the L1 vein. We introduce two methods in which the wing system can be used to study adult mitochondria with particular focus on the axon compartment (see Protocol: Clonal Imaging of Mitochondria in the Dissected Fly Wing [Maddison et al 2022]; Protocol: Live Imaging of Mitochondria in the Intact Fly Wing [Mattedi et al 2022]).…”