In-vivo fast non-linear microscopy reveals intraneuronal transport impairment induced by slight molecular motor imbalances in the brain of zebrafish larvae - Archive ouverte HAL Accéder directement au contenu
Poster De Conférence Année : 2022

In-vivo fast non-linear microscopy reveals intraneuronal transport impairment induced by slight molecular motor imbalances in the brain of zebrafish larvae

Karine Duroure
Filippo del Bene

Résumé

Motor proteins are responsible for the intracellular transport of critical cargoes such as organelles and vesicles along the cytoskeleton. This transport is an vital process, especially in neurons. Axonal transport deficit is found in neurological disorders and is a hallmark of neurodegenerative diseases1. Conventional methods used to measure intraneuronal transport are limited by moderate spatiotemporal resolutions, preventing the observation of events of short duration. We developed a method using photostable optically active nanocrystals (NC) tracer spontaneously internalized in endosomes2. Here we demonstrate the application of this assay to zebrafish (Zf) larvae. We used NC exhibiting large second-order non-linear optical properties, injected in Zf brain. We harnessed these properties combined with fast raster scanning of the infrared laser beam to achieve 20 frames/s rate, allowing us to detect short pausing duration underpinning complex molecular mechanisms otherwise smeared out by low temporal resolution. Using this method in axons of neurons with known polarization, we were able to separate the retrograde from the anterograde phase of motion. We developed a pipeline of video dataset analysis, which extracts the statistical distribution of various transport metrics for both directions, in normal and perturbed situations. To test the sensitivity of our measurement to small perturbations, we modulated the concentration of specific molecular motors, either by applying dynapyrazole3, a retrograde motor dynein inhibiting drug, or by using transgenic Zf engineered with loss-of-function alleles of the anterograde motor protein Kif5aa4. Dynapyrazole induces a reduction 32% of mobile NC, with a 37% reduction of their retrograde run length. In kif5aa mutants the retrograde run length is increased by 46% compared to wildtype. The high sensitivity of our intraneuronal transport measurement assay opens prospects in screening the functional impacts of neurodegenerative disease genetic factors in the animal model of zebrafish larvae for which genetic tools are largely available. References 1.S. Millecamps and J.-P. Julien, Nat. Rev. Neurosci., 2013, 14, 161–176. 2.S. Haziza, et al., Nat. Nanotechnol., 2017, 12, 322–328. 3.J. B. Steinman, et al., Elife, 2017, 6, e25174. 4.T. O. Auer, et al., Elife, 2015, 4, 1–26. 5.S. E. Encalada and L. S. B. B. Goldstein, Annu. Rev. Biophys., 2014, 43, 141–69.

Domaines

Imagerie
Fichier non déposé

Dates et versions

hal-04451566 , version 1 (11-02-2024)

Identifiants

  • HAL Id : hal-04451566 , version 1

Citer

Baptiste Grimaud, Maxence Frétaud, Fériel Terras, Antoine Bénassy, Karine Duroure, et al.. In-vivo fast non-linear microscopy reveals intraneuronal transport impairment induced by slight molecular motor imbalances in the brain of zebrafish larvae. Optogen Workshop 2022, May 2022, Paris, France. ⟨hal-04451566⟩
15 Consultations
0 Téléchargements

Partager

Gmail Facebook X LinkedIn More