Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans

Summary

The paper describes the optimization of fluorescence microscopy acquisition parameters to visualize the axonal transport of endogenous labeled cargos at single-neuron resolution in a living nematode.

Abstract

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).

Introduction

Throughout life, neurons rely on axonal transport to deliver proteins, lipids, and other molecules from the cell body to their final destination in the axon. Consequently, impairment of axonal transport is associated with a loss of neuronal function and is often involved in the pathology of neurodegenerative disorders^1,2. Hence, understanding the mechanisms that underly axonal transport is of great interest.

Several decades of research on axonal transport revealed many important insights into the molecular machinery that mediates this transport, their composition as well as regulat....

Protocol

For a detailed protocol on how to maintain and prepare nematodes for live-cell imaging, refer to the work of S.Niwa ⁷.

1. Worm strain generation

In addition to generating nematode strains, the Caenorhabditis Genetics Center (CGC)⁸ contains a growing collection of nematode strains with endogenously fluorescently tagged proteins that can be directly obtained from their webpage.

1. Choice of the fluorescence labeling strategy
   1. Use the nematode strain MTS1161, which contains the rab-3(ox699) allele⁶, to visual....

Results

Overview of the model system and measurement procedure
To visualize axonal transport of synaptic vesicle precursors, we traced endogenously GFP labeled RAB-3. Here we make use of a recently generated GFP::Flip-on::RAB-3 strain⁶, in which expression of the recombinase Flippase under a cell specific promoter (glr-4p) labels endogenous RAB-3 in the DA9 motor neuron. DA9 is a bipolar motor neuron, with its cell body located in the posterior of the animal on the ventral .......

Discussion

Limitations of the method and alternative methods
In this protocol, we optimized acquisition parameters to visualize the axonal transport of endogenously tagged RAB-3, which is associated with synaptic vesicle precursors. To visualize RAB-3, we made use of a recently published FLIP-on::GFP::RAB-3 strain⁶ and expressed the recombinase Flippase under a cell specific promoter (glr-4p)²⁵. This strategy allows us to label RAB-3 with a single GFP fluorophor.......

Materials

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A9539
Cover slips (22 mm x 22 mm, No1); Gold Seal Cover Glass	Thomas Scientific	6672A14
Levamisole	ChemCruz	sc-205730
Microscope: Nikon Ti2 inverted microscope, Yokogawa CSU-W1 SoRa Scanhead, Hamatsu Orca-Fusion BT sCMOS camera, Nikon CFI Plan Apo lambda 60x 1.4 NA oil immersion objective, Nikon photostimulation scanner at 488nm with an ET525/36 emission filter	Nikon		Spinning Disc Confocal Microscope
NIS-elements AR	Nikon		Software for the Nikon Ti2
Plain precleaned microscopy slides	Thermo Scientific	420-004T
Nematode strain	Identifier	Source
rab-3(ox699[GFP::flip-on::rab-3]) (II); shyIs43(glr-4p::FLP-NLSx2; odr-1p::RFP) (II)	Park et al. (DOI: 10.1016/j.cub.2023.07.052)	MTS1161	Will be deposited at CGC (https://cgc.umn.edu/)