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.

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.
<ol>
	<li>Choice of the ...

Enhancing Axonal Transport Analysis in Living &lt;em&gt;C. elegans&lt;/em&gt; Using GFP-Tagged RAB-3

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+posterior+nervous+system+of+the+nematode+Caenorhabditis+elegans:+serial+reconstruction+of+identified+neurons+and+complete+pattern+of+synaptic+interactions.">The posterior nervous system of the nematode Caenorhabditis elegans: serial reconstruction of identified neurons and complete pattern of synaptic interactions.</a> J Neurosci. 11 (1), 1-22 (1991).</li><li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+ventral+nerve+cord+of+Caenorhabditis+elegans.">The structure of the ventral nerve cord of Caenorhabditis elegans.</a> Philos Trans R Soc Lond B Biol Sci. 275 (938), 327-348 (1976).</li><li>White, J. G., Southgate, E., Thomson, J. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+genome+editing+with+short+single-stranded+and+long,+partially+single-stranded+DNA+donors+in+Caenorhabditis+elegans.">Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans.</a> Genetics. 210 (3), 781-787 (2018).</li><li>Paix, A., Folkmann, A., Rasoloson, D., Seydoux, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency,+homology-directed+genome+editing+in+Caenorhabditis+elegans+using+CRISPR-Cas9+ribonucleoprotein+complexes.">High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes.</a> Genetics. 201 (1), 47-54 (2015).</li><li>Goudeau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-wrmScarlet+and+split-sfGFP:+tools+for+faster,+easier+fluorescent+labeling+of+endogenous+proteins+in+Caenorhabditis+elegans.">Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans.</a> Genetics. 217 (4), (2021).</li><li>Cranfill, P. 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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips (22 mm x 22 mm, No1); Gold Seal Cover Glass</td>
			<td>Thomas Scientific</td>
			<td>6672A14</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>ChemCruz</td>
			<td>sc-205730</td>
			<td></td>
		</tr>
		<tr>
			<td>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</td>
			<td>Nikon</td>
			<td></td>
			<td>Spinning Disc Confocal Microscope</td>
		</tr>
		<tr>
			<td>&#160;NIS-elements AR</td>
			<td>Nikon</td>
			<td></td>
			<td>Software for the Nikon Ti2&#160;</td>
		</tr>
		<tr>
			<td>Plain precleaned microscopy slides</td>
			<td>Thermo Scientific</td>
			<td>420-004T</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode strain</td>
			<td>Identifier</td>
			<td>Source</td>
			<td></td>
		</tr>
		<tr>
			<td>rab-3(ox699[GFP::flip-on::rab-3]) (II); shyIs43(glr-4p::FLP-NLSx2; odr-1p::RFP) (II)</td>
			<td>Park et al. (DOI: 10.1016/j.cub.2023.07.052)</td>
			<td>MTS1161&#160;</td>
			<td>Will be deposited at CGC (https://cgc.umn.edu/)</td>
		</tr>
	</tbody>
</table>

optimizing visualization of axonal transport of endogenous cargo by fluorescence microscopy in living caenorhabditis elegans

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).

Author Spotlight: Advanced Techniques for Visualizing Endogenous Axonal Transport Dynamics

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

Classical start is commonly used for axonal transport proteins for axonal transport visualization, but endogenous cargo behavior differs. Endogenous protein levels are low, making visualization challenging. We offer ideas to optimize the acquisition conditions for better visualization of endogenous GPR3, which labels synaptic vascular precursors.The abundance of cytosolic proteins in the axonal transport field, makes it difficult to visualize their dynamics, using conventional fluorescence microscopy. This protocol suggests using ablation step, along with temporally controlled labeling approaches to overcome these challenges. We have recently been able to visualize the axonal transport of the endogenous spectry.By optimizing the acquisition parameters described in this protocol, paired our temporal control labeling approach. This allowed us to identify the first components of the machinery that mediate the transport.

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 ...

Watch this Scientific Journal Video about Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans at JoVE.com

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.

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. ...

optimizing-visualization-axonal-transport-endogenous-cargo

Research

JoVE Journal

Biology

Handling and Preparation of Caenorhabditis elegans for Live Imaging

To begin, take out the nematode growth medium plates with Caenorhabditis elegans grown on a lawn of bacteria. Using a platinum wire pick, transfer worms to a droplet of 0.5 millimolar levamisole and incubate them for 20 minutes. Pipette 12 microliter M9 medium to a 10%agarose patch on a glass slide.Using a hair pick, mount 10 to 20 nematodes from the levamisole droplet into the M9 droplet on the agarose patch. Place a cover slip on top of the droplet and rotate it by 45 degrees to image transport in the DA9 axon. Afterward, seal the space between the cover slip and the glass slide with a viscous gel-like petrolatum.

Visualizing Axonal Transport in a Live Caenorhabditis elegans Worm

To begin, take the caenorhabditis elegans slide prepared for imaging. Observe the slide under 4x to 20x magnification lens to locate nematodes. Remember their positions, and then switch to a 63x magnification lens for detailed imaging.Capture an initial image to evaluate acquisition parameters. Monitor signal intensities using the intensity histogram, and increase excitation laser intensity if necessary. Next, using a 488 nanometer laser, bleach the region of the axon to be analyzed.Compare the signal intensity before and after the bleaching step to determine the bleaching efficiency. Use two by two binning to enhance the signal intensity of individual transport events. Enhance the exposure time and use a temporal resolution of 300 to 700 milliseconds between consecutive time points to follow Rab3 transport events.Record a time lapse of one to three minutes based on the signal intensity and frequency of transport events.

Generating a Kymograph and Measuring Individual Transport Events in a Live Caenorhabditis elegans Worm

To begin, record a timelapse of endogenous GFP Rab3 in the asynaptic zone of the DA9 axon in a live Caenorhabditis elegans. Import the timelapse data file into Fiji. Then check for movement or slight drift of the animal or axon segment during image acquisition.Use the StackReg plugin with a rigid body transformation option to correct for any movement or drifting. Employ the segmented line tool and adjust the line width to match the axon diameter. Double click with the left mouse button on the segmented line icon to draw a line along the axon segment for analysis.Now using the Fiji plugin KymoResliceWide, generate the kymograph, setting the maximum intensity value across the width of the line in its parameter settings. Using the straight line tool, trace transport events in the kymograph. trace individual transport events and save each line to the ROI manager.In Fiji, select measurement parameters such as area, bounding rectangle, mean gray value, and Feret's diameter. Then paste the results table into a spreadsheet for calculation. Multiply the width by the resolution of the camera to get the run length in micrometers.To determine the duration of a movement event, multiply the height by the acquisition time between consecutive time points. Calculate pausing time as the run duration between two consecutive movements where velocity is zero. Normalize the total events to the total length of the kymograph for event number per minute and axon length segment.Using the Ferret angle tool, categorize events into anterograde and retrograde transport. For kymographs generated by drawing the segmented line from proximal to distal, use the Feret angle to determine the directionality of each event.