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

Published: February 16, 2024

doi:

¹Department of Neuroscience,Yale School of Medicine

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 disorders¹^,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 regulatory mechanisms. Long-range axonal transport occurs on the microtubule cytoskeleton, which consists of partially overlapping microtubule polymers that are typically oriented with their plus end out in axons³. Consequently, anterograde transport is mediated by motor proteins that walk to the plus end of microtubules, kinesins, whereas retrograde transport depends on the minus end directed dynein motor. Although many aspects of transport have been revealed, for many axonal proteins it still remains unclear, how they are loaded into the transport machinery, how individual transport packages are organized, and how this transport is regulated³.

Axonal transport was initially studied in radio-labeling experiments, in which radiolabeled amino acids were injected into the somatic compartment, where they were incorporated into nascent endogenous proteins and could be traced over time in the axonal compartment by autoradiography⁴. Although radiolabeling experiments allowed the study of axonal transport of endogenous proteins in vivo, it does not allow for the direct follow-up of the behavior of individual cargo to get mechanistic insights⁴. This limitation was overcome with the use of fluorescence microscopy. However, axonal transport is often not visualized on endogenous proteins but instead by expression of a fluorescent labeled copy. Especially for low expressed proteins, overexpression provides higher signal intensities which make visualization, preferably with single neuron resolution, possible. Moreover, ectopic expression of the fluorescent tagged protein circumvents the need and challenges of genome editing. Conversely, it has been argued that the behavior of ectopically expressed cargo may differ from the behavior of the endogenous cargo⁵.

Recent improvements in genome editing made endogenous labeling strategies easier accessible. Hence, a lower signal intensity has become the major limitation to study axonal transport of a cargo by ectopic expression instead of endogenous labeling. Careful considerations in the endogenous labeling strategy paired with an optimization of the acquisition conditions can overcome this challenge.

Nematodes provide an excellent research model to study axonal transport in vivo due to their transparency and ease in genetic manipulations. In this protocol, we describe a research strategy to visualize axonal transport of endogenous proteins with single neuron resolution in living Caenorhabditis elegans. We visualize the axonal transport of synaptic vesicle precursors by using a strain generated by the Jorgensen Lab⁶, in which the vesicle associated RAB GTPase, RAB3, is endogenously labeled with GFP, in the motor neuron DA9. By asking how small adaptations in different acquisition parameters and photobleaching can improve the visualization of individual transport events, the protocol provides ideas on how to optimize imaging conditions.

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. Choice of the …

Representative 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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to thank the Yogev and Hammarlund labs for technical assistance, feedback, and discussions. We would like to especially thank Grace Swaim for guidance in live cell imaging and Grace and Brian Swaim for initially establishing the manual kymograph analysis in the lab. OG is supported by a Walter-Benjamin Scholarship funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) -Project# 465611822. SY is funded by the NIH grant R35-GM131744.

Materials

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/)

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