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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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 .......

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.......

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

NameCompanyCatalog NumberComments
AgaroseSigma-AldrichA9539
Cover slips (22 mm x 22 mm, No1); Gold Seal Cover GlassThomas Scientific6672A14
LevamisoleChemCruzsc-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 filterNikonSpinning Disc Confocal Microscope
 NIS-elements ARNikonSoftware for the Nikon Ti2 
Plain precleaned microscopy slidesThermo Scientific420-004T
Nematode strainIdentifierSource
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/)

References

  1. Millecamps, S., Julien, J. P. Axonal transport deficits and neurodegenerative diseases. Nat Rev Neurosci. 14 (3), 161-176 (2013).
  2. Brady, S. T., Morfini, G. A. Regulation of motor proteins, axonal t....

Explore More Articles

Axonal TransportEndogenous CargoFluorescence MicroscopyCaenorhabditis ElegansCRISPR Cas9Synaptic Vesicle PrecursorsRAB 3Acquisition ParametersBleachingOptimization

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