Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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, respectively^1,2.

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 IFT^7,8,9. To observe IFT, Chlamydomonas has been widely used as a model organism^5,6. 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 time^12,13. 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 lethal^7,9,14. 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.

Protokół

1. Sample Preparation

1. 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 strains are used in this protocol.
   NOTE: Either extrachromosomal array, genomic integration, or even GFP knock-in works. To reduce background signals, use cell-specific promoters, rather than pan-neuronal promoters.
   NOTE: Maintenance of worms is described in other protocols²⁰. Strains were maintained on lawns of Escherischia coli OP50 feeder on nematode growth medium (NGM) plates (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 on 60 mm diameter plastic dishes) at 20 °C.
2. Grow worms at 20 °C on NGM plates to any life cycle stage of the worms.
   NOTE: One can observe both axonal transport and IFT at any stage. Late L4 to young adult is a good positive control because multiple publications have used these stages and visualized trafficking events^14,21,22,23.

2. Preparation of 10% Agarose

NOTE: Many vendors provide similar products such as agar, agar powders, and agarose. Use electrophoresis grade agarose (gel strength >1200 g/cm²). Cheap agar powders do not work because the resulting gel is not strong enough to immobilize worms.

1. Mix 0.4 g agarose in 4 mL distilled water (DW) in a glass tube (1.5 cm x 10.5 cm). Put a lid on the glass tube to avoid evaporation.
2. Put the glass tube on a 95 °C heat block for 90 min. In addition, prepare another glass tube (1.5 cm x 10.5 cm) filled with DW and keep it at 95 °C. Put a Pasteur pipet in the 95 °C DW (Figure 1A). A lot of small bubbles will be seen in the agarose tube and the solution should be turbid. Leave it until the solution becomes clear. Then, mix by pipetting up and down until the agarose solution becomes homogeneous.
   NOTE: Microwaves cannot be used to prepare 10% agarose solution when made from DW and agarose. Small bubbles caused by microwaves prevent agarose melting. However, solidified 10% agarose gel can easily be melted again using a microwave. Glass tubes containing 10% agarose gel can be prepared ahead of time and stored at 4 °C for at least 6 months. If doing so, melt the stock using a microwave when needed and start from step 3 in this protocol.
   NOTE: Agarose gradually loses the ability to solidify if incubated at 95 °C for a long time or after repeated solidification and melting. If this happens, prepare new 10% agarose.

3. Preparation of Agarose Pad

1. Using the warmed Pasteur pipet prepared in step 2.2, place 2 drops of 10% agarose on a 76 x 22 mm slide.
2. Quickly cover with a second 76 x 22 mm² slide before the agarose drop solidifies as shown in Figure 1B, and push down on the second slide to flatten the agarose solution. The thickness should be 0.5-1 mm.
3. Quickly flush the Pasteur pipette by pipetting 95 °C DW up and down until the agarose solution is washed out. Otherwise, the pipet will become clogged by agarose gel. Leave the pipette standing in the 95 °C DW.
4. Wait for 1 min. The agarose pad forms and becomes cloudy. Then, slide the slides apart by hand (Figure 1C).

4. Mounting of Worms

NOTE: Even small amounts of worm movement prevent good observation. Levamisole has traditionally been used to prevent worm movement on the agarose pad^24,25. However, Levamisole inhibits neuronal receptors in C. elegans, and therefore may affect trafficking events in neurons¹⁵. Using polystyrene microbeads described hereis a good alternative¹⁷.

1. Under a stereo microscope equipped with a slidable mirror transillumination, transfer ~30 transgenic worms expressing the appropriate markers to a new NGM plate without bacteria using a platinum wire pick.
   NOTE: A platinum wire pick is made of platinum wire and Pasteur pipette (5 inches), and the tip of the platinum wire was flattened. The preparation of the platinum wire picks and handling of worms with these are described in the Wormbook (http://www.wormbook.org).
2. Leave the plate for 1 min. Bacteria are automatically removed from the surfaces of worms as the worms move on NGM agarose gel without bacteria.
3. Put a 0.5-1.0 µL drop of 2.6% 100 nm diameter polystyrene microbeads in water onto the agar pad (Figure 1D). Transfer 10-20 worms from the bacteria-free NGM plate to the microbeads. Drop and spread the worms using the platinum wire pick to avoid the overlapping of worm bodies (Figure 1E).
4. Apply a 22 x 40 mm² coverslip (Figure 1F). Push gently to remove air bubbles, but avoid crushing worms. The sample is now ready for imaging.
   NOTE: Because no anesthesia is administered, worms are fixed only by the frictional force generated by the agar pad, polystyrene microbeads, and coverslip¹⁷. The presence of feeder bacteria and/or too much solution will make the frictional force weaker.
   NOTE: Different sizes of coverslips (e.g., 22 x 22 mm², 18 x 18 mm²) work. However, larger coverslips can prevent immersion water or oil from the objective lens leaking into samples during the observation.

5. Observation

NOTE: Appropriate imaging parameters (laser power, gain, binning, etc.) will differ for each microscope system and camera. Here, a widefield microscope equipped with a spinning disk confocal scanner and a digital CCD camera is used.

1. Set the temperature control of the stage to 20 °C, or adjust the room temperature to 20 °C.
2. Put the sample slide on the microscope stage. Use 100x (numerical aperture = 1.3 or better) objective lens.
3. Look for the areas indicated in Figure 2A (for wyIs251 and axonal transport) or Figure 2B (for mnIs17 and IFT) as positive controls. Other areas can be analyzed as well²².
4. Set parameters (CCD Gain = 200, Binning = 1 or 2, laser power at 488 nm = 1.0-1.3 mW). Perform time lapse recording at 4 frames/s for up to 1 min. Save files as mutli-TIFF format.
   NOTE: If GFP signals disappear and it is difficult to continue to observe GFP::RAB-3 or OSM-6::GFP for 30 s, the laser power is too strong. In that case, reduce the laser power. Conversely, if no vesicular movement is recorded, the laser power is too weak. In that case, increase the laser power.
5. Observe a different worm and repeat 5.3 and 5.4. The observations can last for up to 20 min, but each worm should be recorded only once to avoid the effects of photo damage.
   NOTE: Worms have been observed for at least 20 min without significant defects^7,27,28. Longer observation may be possible but haven't been tested yet. A clear sign of neuronal death is the change of neuronal morphology such as neurite swelling. As weak GFP signals can be seen in axons and dendrites in both wyIs251 and mnIs17, neurite morphology can be easily checked by just looking at axons or dendrites. Neuronal morphology is shown in Figure 2.
6. Generate movie files.
   1. Open the multi-TIFF file using Fiji software. Go to File>Open then select the multi-TIFF file saved in step 5.4.
      NOTE: Fiji can be downloaded at jttps://fiji.sc. Image J and plugins can also be used to generate kymographs. If doing so, follow the relevant instructions for the chosen plugin. 
   2. Click the "Rectangular selection" button (Figure 3A, arrow) and select the area of interest by drawing a rectangle with a computer mouse (yellow rectangle in Figure 3A). Go to Image>Stacks>Tools>Make Substacks and select the slice numbers that are included in the movie. A substack is generated.
   3. Activate the substack window by clicking and go to Image>Adjust>Brightness/Contrast. A window to adjust the brightness and contrast shows up. In the window, slide the top bar (Minimum) to the right so that the background signal disappears and the second top bar (Maximum) to the left so that the moving vesicles and particles can be seen quite well over the background.
   4. Go to Image>Stacks>Time Stamper. As the movie is recorded at 4 frames/s in step 5.4, the time interval is 0.25 s. Thus, type "0.25" in Interval.
   5. Generate a movie file by selecting Save as>AVI from the File menu (Movie 1 and 2).
      NOTE: By using appropriate plugins, you can save the file as other formats such as "mpeg4" or "mov" files.
7. Generate kymographs.
   1. Open the original movie files using FIji software again and repeat steps 5.6.2 and 5.6.3 to adjust the brightness and contrast.
   2. Click "segmented line" (Figure 3B, arrow). Using a mouse, draw a segmented line along the cilia or axon (yellow line, in Figure 3B).
   3. Go to Analyze>Multi Kymograph>Multi Kymograph (Figure 3C). Line width window appears (Figure 3D). Type "3" in the Linewidth window and Click OK (Figure 3D).
   4. Kymograph window is created. Save the image in TIFF or JPEG format.

Wyniki

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 μm/s, respectively²². The number of moving vesicles is about 0.03 and 0.018 per μm of axon per s. Thus, for a 30 s observati...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Slideglass (76  x 26 mm)	Matsunami	S1111
Coverglass (22  x 40 mm)	Matsunami	C024401
Agarose	Wako	318-01195
Polystylene microbeads 0.1 micron	Polysciences	#00876
Heat block	TAITEC	0063288-000	CTU-mini
Microscope	Olympus	IX-71	widefield microscope
Spinning disk Scanner	Yokogawa	CSU-X1	spinning disc confocal scanner
Digital CCD camera	Hamamatsu Photonics	C10600-10B	ORCA-R2 degital CCD camera
Objective lens (x100, NA1.4)	Olympus	UPLSAPO 100XO
Pasteur pipette (5 inch)	IWAKI	IK-PAS-5P
Glass tube (1.5 cm diameter x 10.5 cm)	IWAKI	9820TST15-105NP
TV9211: wyIs251	Laboratory of Kang Shen	N/A
OTL11: mnIs17	Ref. 27, 29	N/A	SP2101 was backcrossed with wild type for 6 times
Stereo microscope	Carl Zeiss	435064-9000-000	STEMI 508
Mirror transillumination unit	Carl Zeiss	435425-9010-000
platinum wire (0.2 mm)	Nilaco Corporation	m78483501
60 mm plastic dish	Falcon	#351007
Fiji	N/A	N/A	https://fiji.sc/
nematode growth medium (NGM)			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