Name: immobilization of caenorhabditis elegans to analyze intracellular transport in neurons
Uploaded: 2017-10-18T15:00:00
Description: Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

The author deeply thanks Dr. Asako Sugimoto (Tohoku University) for her helpful discussion. wyIs251 was a generous gift from Dr. Kang Shen (Stanford University). mnIs17 was provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was supported by JSPS KAKENHI grant #17H05010 and #16H06536 and Daiichi Sankyo foundation, Brain Science Foundation and Naito Foundation.

1. Sample Preparation
<ol>
	<li>Generate a transgenic C. elegans strain of interest using transgenic methodologies18. Alternatively, obtain appropriate strains from the Caenorhabditis Genetics Center (CGC). To visualize axonal transport of synaptic vesicle precursors, use the transgenic line wyIs251 [Pmig-13::gfp::rab-3]7. To observe IFT, obtain the transgenic line mnIs17 [osm-6::gfp]19. These s...

<ol>
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Limitation with respect to existing methods 
The method described here is optimized to observe fast events such as axonal transport and IFT. Thus, immobilization is more prioritized than longer incubation. While we have been able to observe trafficking events for at least 20 min without significant perturbation, this method may not be always suitable to observe slow events requiring longer observations, such as axon elongation and cell migration. For longer observations, one needs to optimize the co...

Live cell imaging is an essential tool for analyzing intracellular transport. In neuronal cell biology, analyses of axonal transport with live cell imaging are essential for understanding neuronal function and morphogenesis1. Defects in axonal transport underlie several neurodegenerative disorders2. Kinesin superfamily proteins and dynein carry out axonal transport anterogradely and retrogradely, respectively1,2.
Cilia are another cellular compartment in which the microtubule network and trafficking machinery are highly developed3. Protein synthesis machinery is not localized in cilia, which means that ciliary proteins must be transported from the cytoplasm to the cilia tips. Cilia-specific kinesin and dynein, called kinesin-2 and cytoplasmic dynein-2, respectively, transport the components of cilia4, in a phenomenon called intraflagellar transport (IFT)5. Impairment of IFT causes a spectrum of diseases called ciliopathies6. Thus, analysis of the IFT mechanism by live cell imaging is required to understand basic mechanisms of ciliary formation and pathogenesis.
Caenorhabditis elegans (C. elegans) is a good model to study axonal transport and IFT7,8,9. To observe IFT, Chlamydomonas has been widely used as a model organism5,6. As Chlamydomonas is a unicellular organism, the relationship of IFT with aging, neuronal function, and behavior would be difficult to analyze. In addition, essential genetic techniques such as CRISPR/Cas9 have not been applied to Chlamydomonas. In higher model organisms, such as mice and Drosophila, disruption of axonal transport and IFT often causes lethal phenotypes because axonal transport and IFT are essential for the morphogenesis and homeostasis of the animals 10,11. In the case of mice, cell culture and transfection is generally needed to observe axonal transport and IFT, which requires many skills and extensive time12,13. In addition, a lot of important physiological context may be lost in cultured cells and cell lines. Because the nervous system is not essential for the survival of the worms, C. elegans mutants in which axonal transport or IFT are disrupted are often not lethal7,9,14. Axonal transport and IFT can be directly observed in vivo without dissection because C. elegans is transparent and it is therefore easy to observe GFP-tagged markers.
There are several protocols to immobilize C. elegans, such as using a microfluidic device15, agarose pads with anesthesia16, or microbeads17. The inclusion of anesthesia may inhibit the trafficking events in neurons15. A clear drawback of the microfluidic-device method is that preparing a microfluidic device is not always easy. Instead, immobilization by agarose pads and microbeads is a convenient and easy way to perform time lapse imaging in C. elegans. Here, I describe this basic protocol to immobilize C. elegans and visualize axonal transport and IFT in vivo in C. elegans. Compared to the other methods, the method described here does not require special equipment and is much cheaper and easier to perform.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Slideglass (76&#160; x 26 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">S1111</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Coverglass (22&#160; x 40 mm)</td>
			<td style="width:167px;">Matsunami</td>
			<td style="width:139px;">C024401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Agarose</td>
			<td style="width:167px;">Wako</td>
			<td style="width:139px;">318-01195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Polystylene microbeads 0.1 micron</td>
			<td style="width:167px;">Polysciences</td>
			<td style="width:139px;">#00876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Heat block</td>
			<td style="width:167px;">TAITEC</td>
			<td style="width:139px;">0063288-000</td>
			<td>CTU-mini</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">IX-71</td>
			<td>widefield microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Spinning disk Scanner</td>
			<td style="width:167px;">Yokogawa</td>
			<td style="width:139px;">CSU-X1</td>
			<td>spinning disc confocal scanner&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Digital CCD camera</td>
			<td style="width:167px;">Hamamatsu Photonics</td>
			<td style="width:139px;">C10600-10B</td>
			<td>ORCA-R2 degital CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Objective lens (x100, NA1.4)</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:139px;">UPLSAPO 100XO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Pasteur pipette (5 inch)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">IK-PAS-5P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Glass tube (1.5 cm diameter x 10.5 cm)</td>
			<td style="width:167px;">IWAKI</td>
			<td style="width:139px;">9820TST15-105NP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">TV9211: wyIs251</td>
			<td style="width:167px;">Laboratory of Kang Shen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">OTL11: mnIs17</td>
			<td style="width:167px;">Ref. 27, 29</td>
			<td style="width:139px;">N/A</td>
			<td>SP2101 was backcrossed with wild type for 6 times</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Stereo microscope</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td style="width:139px;">435064-9000-000</td>
			<td>STEMI 508&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mirror transillumination unit</td>
			<td style="width:167px;">Carl Zeiss</td>
			<td>435425-9010-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">platinum wire (0.2 mm)</td>
			<td style="width:167px;">Nilaco Corporation</td>
			<td style="width:139px;">m78483501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm plastic dish</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:139px;">#351007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Fiji</td>
			<td style="width:167px;">N/A</td>
			<td style="width:139px;">N/A</td>
			<td>https://fiji.sc/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">nematode growth medium (NGM)&#160;&#160;</td>
			<td style="width:167px;"></td>
			<td style="width:139px;"></td>
			<td>1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) Peptone, 1 mM CaCl2, 5 mg/mL Cholesterol, 25 mM KH2PO4, 1 mM MgSO4&#160;</td>
		</tr>
	</tbody>
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immobilization of caenorhabditis elegans to analyze intracellular transport in neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Axonal transport in DA9 neuron 
Using the wyIs251 line, both the anterograde and retrograde axonal transport of GFP::RAB-3 can be simultaneously recorded in the DA9 motor neuron. The average speed of anterograde and retrograde transport in the proximal dorsal axon of the DA9 neuron is about 1.8 and 2.6 &#956;m/s, respectively22. The number of moving vesicles is about 0.03 and 0.018 per &#956;m of axon per s. Thus, for a 30 s observati...

Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

The overall goal of this time lapse recording is to observe the intracellular transport events in Caenorhabditis elegans neurons.This method can help answer key questions in the cell biology field such as axonal transport and interflagellar transport.The main advantage of this technique is that one can observe intracellular transport in C.Elegans without dissection.Start this experiment by growing transgenic C.elegans strain of interest at 20

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Developmental Biology

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