We thank Dr. Bing-Yan Wang, Dr. Gena Tribble (The University of Texas, School of Dentistry), Dr. Richard Lamont (University of Louisville, School of Dentistry), and Dr. Samuel Shelburne (MD Anderson Cancer Center) for providing laboratory and clinical strains of the mitis group streptococci. We also thank Dr. Keith Blackwell (Department of Genetics, Harvard Medical School) for the C. elegans strains. Finally, we thank Dr. Danielle Garsin and her lab (The University of Texas, McGovern Medical School) for providing reagents and worm strains to conduct the study. Some worm strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

1. Preparation of THY (Todd-Hewitt Yeast Extract) Agar Plates
<ol>
	<li>For 1 L of media, add 30 g of Todd-Hewitt powder, 2 g of yeast extract and 20 g of agar to a 2 L Erlenmeyer flask. Add 970 mL of deionized water to the contents of the flask and include a stir bar. Autoclave the media at a temperature of 121 &#176;C and pressure of 15 lb/inch2 for 30 min. Thereafter, set the media on a stir plate and allow for cooling with gentle stirring.</li>
	<li>Pour the media into appropriately sized sterile Petri dishes (100 mm x 15 mm dishes for growth and maintenance of bacteria, 35 mm x 10 mm dishes for killing assays) under a laminar flow. Allow the media to dry for 2 h under the laminar hood. Thereafter, the plates can be stored at 4 &#176;C for 1 month.</li>
</ol>
2. Preparation of Nematode Growth Medium (NGM) and RNAi Feeding Plates (NGM RNAi)
<ol>
	<li>Using a stir bar, dissolve 2.5 g of peptone and 3 g of NaCl in 970 mL of deionized water in a 2 L Erlenmeyer flask. Add 20 g of agar to the media. Autoclave the media at a temperature of 121 &#176;C and pressure of 15 lb/inch2 for 30 min. Set the media on a stir plate and allow for cooling with gentle stirring.</li>
	<li>Add the following solutions to the media for preparation of NGM plates: 25 mL of 1 M potassium phosphate buffer (pH = 6.0), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 1 mL of (5 mg/mL in 95% ethanol) cholesterol, 1 mL of (10% v/w in ethanol) nystatin, and 1 mL of 25 mg/mL streptomycin.</li>
	<li>Add the following solutions to the media for preparation of NGM RNAi feeding plates: 25 mL of 1 M potassium phosphate buffer (pH = 6.0), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 1 mL of (5 mg/mL in 95% ethanol) cholesterol, 1 mL of (10% v/w in ethanol) nystatin, 1 mL of 50 mg/mL carbenicillin, and 1 mL of IM IPTG.</li>
	<li>Pour the media into 60 mm x 15 mm sterile Petri dishes under laminar flow. Allow the media to dry for 2 h under the laminar hood. Subsequently, plates can be stored at 4 &#176;C for 1 month.</li>
</ol>
3. Maintenance of C. elegans
<ol>
	<li>Seed the NGM plates by spotting 50 &#181;L of overnight grown E. coli OP50 in the center of the plates. The E. coli culture is prepared previously in Luria-Bertani (LB) media and stored at 4 &#176;C for several months. Cover the plates and allow them to dry for 24 h under a laminar hood and thereafter store the plates in polystyrene container.</li>
	<li>Under a dissecting microscope, pick up 10 to 12 gravid adults using a sterile worm pick and transfer the worms to an E. coli OP50 seeded NGM plate. Incubate the plates at 20 &#176;C overnight.</li>
	<li>The next day, remove the adults using a sterile worm pick and allow the embryos to develop to L4 larvae at 20 &#176;C (~2.5 days).</li>
</ol>
4. Preparation of Age Synchronous Population of Worms
<ol>
	<li>Wash gravid adults from two to four NGM plates using M9W and collect them in a 15 mL conical tube.
	<ol>
		<li>To prepare M9W: combine 3 g of NaCl, 6 g of Na2HPO4, and 3 g of KH2PO4 and dissolve in a final volume of 1 L of deionized water. Autoclave the solution and add 1 mL of 1 M MgSO4.</li>
	</ol>
	</li>
	<li>Spin the tube at 450 x g for 1 min, then decant the supernatant while ensuring that the worm pellet stays intact.</li>
	<li>Add 400 &#181;L of 8.25% sodium hypochlorite (household bleach) and 100 &#181;L of 5 N NaOH to prepare the worm lysis solution. Add the lysis solution to the worm pellet and mix the contents by flicking the tube until 70% of the adult worms are lysed. Periodically observe the contents of the tube under a dissecting microscope to ensure there is no overbleaching of the eggs.</li>
	<li>Dilute the bleach mix by adding 10 mL of M9W to the contents of the conical tube.</li>
	<li>Spin the tube at 450 x g for 1 min. Decant supernatant, then add 10 mL of M9W.</li>
	<li>Repeat step 4.5 two more times.</li>
	<li>Resuspend the resulting egg pellet in 3-5 mL of M9W. Place the tube on a tube rotator. Allow the tubes to rotate at a speed of 18 rpm at room temperature (RT) overnight.</li>
	<li>The next day, spin the tube at 450 x g for 1 min to pellet the L1 larvae. Remove most of M9W by aspiration, leaving behind ~250 &#181;L of liquid in the tube. Resuspend the L1 larvae and place three 5 &#181;L drops of the worm suspension onto a Petri dish lid, then estimate the number of worms per &#181;L using a dissecting microscope.</li>
</ol>
5. Induction of RNAi in Worms
<ol>
	<li>Using a sterile loop or pick, streak out the desired strains of RNAi-containing E. coli from frozen stocks (Ahringer and Vidal libraries) onto 100 mm x 15 mm LB agar plates containing 50 &#181;g/mL carbenicillin and 15 &#181;g/mL of tetracycline. Incubate the plates at 37 &#176;C for 24 h.</li>
	<li>Pick and inoculate an isolated colony from the desired RNAi strain into sterile 15 mL conical tubes containing 2 mL of LB supplemented with 50 &#181;g/mL carbenicillin. Incubate the tubes at 37 &#176;C for 16 h in an orbital shaker at 150 rpm.</li>
	<li>The next day, spread 150 &#181;L of overnight grown culture onto 65 mm x 15 mm NGM RNAi feeding plates using a sterile spreader. Incubate the plates at 37 &#176;C for 24 h.</li>
	<li>Allow the plates to cool to RT after incubation at 37 &#176;C. Add an appropriate volume of M9W containing ~200 L1 larvae (obtained from the age synchronous population of worms steps) to the E. coli seeded NGM RNAi feeding plates. Incubate the plates at 20 &#176;C until the larvae reach the L4 stage (~2.5 days).</li>
</ol>
6. Preparation of Mitis Group Streptococci for Infection
<ol>
	<li>Streak out desired strains of streptococci on the 100 mm x 15 mm THY agar (if plates were stored at 4 &#176;C, pre-warm the plates to 37 &#176;C before streaking the respective strains), then incubate the plates at 37 &#176;C overnight (~18 h) in a candle jar providing a microaerophilic environment for the growth of the streptococci (the streaked plates can be stored for a week at 4 &#176;C).</li>
	<li>To propagate clinical isolates of streptococci, use tryptic soy blood agar. Incubate the plates at 37 &#176;C in a candle jar overnight (~18 h).</li>
	<li>The next day, remove the plates from the candle jar and pick isolated colonies using a sterile loop. Inoculate 15 mL sterile conical tubes containing 2 mL of THY broth. To propagate clinical isolates of streptococci, supplement the THY broth with 5% v/v of sheep blood. Close the caps tight and incubate the tubes at 37 &#176;C under static conditions.</li>
</ol>
7. Survival Assays
NOTE: The steps involved in this assay are depicted in Figure 1. To demonstrate that the H2O2 derived by the mitis group is responsible for the killing of the worm, supplement the media with catalase, or the mutant strain &#916;spxB and complement strain &#916;spxB;spxB+ of S. gordonii can be used. SpxB encodes for a pyruvate oxidase, which is responsible for the production of H2O2 in the mitis group.
<ol>
	<li>Pre-warm 35 mm x 10 mm THY plates to 37 &#176;C. Add 80 &#181;L of overnight grown cultures of the desired strains of streptococci and spread the bacteria completely across the agar surface using a sterile spreader. Incubate the plates at 37 &#176;C in a candle jar overnight (~18 h). As a control, seed two 35 mm x 10 mm NGM plates with 80 &#181;L of overnight grown cultures of E. coli OP50. Incubate the plates at 37 &#176;C overnight.</li>
	<li>To confirm that the H2O2 produced by the mitis group is responsible for killing of the worms, add 50 &#181;L containing 1,000 units of catalase c onto the THY plate. Spread the catalase solution using a sterile spreader and allow the plates to dry in the laminar flow for 30 min. Seed the plates thereafter with the respective streptococcus strains as described in step 7.1.</li>
	<li>The next day, remove the plates from the candle jar and allow the plates to cool to RT for 10-15 min. Using a sterile worm pick, transfer 30 L4 larvae from the NGM or NGM RNAi feeding plates to the streptococcus seeded THY plates. Use two seeded THY plates with a total of 60 worms per strain of streptococcus. Incubate the plates at 25 &#176;C.</li>
	<li>Using a dissecting microscope, count the number of live and dead L4 larvae on each plate at several timepoints. Initially, score the worms as dead or live every 30 min. Thereafter, when worms rapidly start to die, score them at 15 min intervals. Use the sterile worm pick to gently prod the worms and determine if they are dead or alive. A worm is considered dead if there is no movement in response to the prodding.</li>
	<li>The assay will take 5-6 h to complete. Repeat the experiment two more times. After completion of the assay, pool the data from the two plates. Input the data of each group, compare the survival curves, and perform Kaplan-Meier survival analysis using statistical software.</li>
</ol>
8. Preparation of Agarose Pads for Microscopy
<ol>
	<li>Dissolve 2% w/v of agarose in deionized water by heating the solution in a microwave. A volume of 5 mL of solution is adequate to prepare 20 slides.</li>
	<li>Stick lab tape lengthwise along two glass slides. This will determine the thickness of the agarose pads. Place a clean glass slide between the two taped slides.</li>
	<li>Place 100 &#181;L of molten agarose on the center of the clean slide. Immediately place another clean glass on top of the molten agarose and gently press down to make a pad. Allow the agarose to solidify and subsequently remove the top slide. The agarose pad is ready for use.</li>
</ol>
9. Observation of SKN-1 Localization in Response to Streptococcus Infection
NOTE: The steps involved in this assay are depicted in Figure 2. Localization of SKN-1 was determined using the SKN-1B/C::GFP transgenic worm strain. To demonstrate localization of SKN-1 due to the production of H2O2 by the mitis group, wild-type (WT), &#916;spxB, and the complement strain&#160;&#916;spxB;spxB+ of S. gordonii were used. Furthermore, the transgenic reporter strain SKN-1B/C::GFP and RNAi interference technique were used to demonstrate that components of the p38 MAPK pathway regulate the localization of SKN-1.
<ol>
	<li>Pre-warm 35 mm x 10 mm THY plates to 37 &#176;C. Add 80 &#181;L of overnight grown cultures of the desired strains of streptococcus and spread the bacteria completely across the agar surface using a sterile spreader. Incubate the plates at 37 &#176;C in a candle jar overnight (~18 h). As a control, seed three 35 mm x 10 mm NGM plates with 80 &#181;L of overnight grown cultures of E. coli OP50. Incubate these plates at 37 &#176;C for ~18 h.</li>
	<li>The next day, remove plates from the candle jar and allow the plates to cool to RT for 10-15 min. Wash L4 larvae using M9W from NGM and NGM RNAi feeding plates. Collect the worms in 15 mL conical tubes.</li>
	<li>Spin the tubes at 450 x g for 1 min. Decant the supernatant and add 10 mL of M9W.</li>
	<li>Repeat step 9.3 three more times.</li>
	<li>Resuspend the worms in ~250 &#181;L of M9W and place three 5 &#181;L drops of the worm suspension onto a clean Petri dish lid and estimate the number of worms per &#181;L using a dissecting microscope.</li>
	<li>Add ~100 L4 larvae to each THY streptococcus seeded and NGM E. coli seeded plates. Use three plates per strain of bacteria. Incubate the plates for 2 to 3 h at 25 &#176;C.</li>
	<li>Thereafter, remove the plates from the incubator, wash them with M9W, and collect the worms in 15 mL conical tubes.</li>
	<li>Wash the worms 3x as described in steps 9.3.</li>
	<li>Remove most of the M9W by aspiration and add 500 &#181;L of M9W containing 2 mM sodium azide or 2 mM tetramisole hydrochloride to the worm pellet. This will anesthetize the worms, ensuring that no movement occurs when imaged under the microscope. 
	CAUTION: Use personal protective equipment (PPE) when handling sodium azide. Prepare the azide solution under a chemical hood.</li>
	<li>Incubate the worm pellets at RT for 15 min. Then, spot 15 &#181;L of the worm suspension onto a prepared agarose pad. Gently place a no. 1.5 coverslip over the agarose pad containing the anesthetized worms.</li>
	<li>Using a fluorescent microscope, visualize the localization of SKN-1 utilizing FITC and DAPI filters.Image worms at 10x and 20x magnifications.</li>
	<li>Score the worms based on the level of localization of SKN-1. No nuclear localization, localization of SKN-1B/C::GFP in the anterior or posterior of the worm, and nuclear localization of SKN-1B/C::GFP in all intestinal cells are categorized as low, medium, and high levels of localization, respectively.</li>
	<li>After scoring the fluorescent micrographs, determine the statistical differences by chi- squared and Fisher&#39;s exact tests using statistical software.</li>
</ol>

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The authors declare that they have no competing financial interests.

The methods described can be used for other pathogenic bacteria such as Enterococcus faecium, which also produces H2O2 grown under anaerobic or microaerophilic conditions26. Typically, for most pathogenic organisms, it takes several days to weeks to complete the survival assays. However, due to the robust production of H2O2 by members of the mitis group, these assays could be completed within 5-6 h under the conditions described. This ensures th...

Mitis group streptococci are human commensals of the oropharyngeal cavity1. However, these organisms can escape this niche and cause a variety of invasive diseases2. The infections caused by these microorganisms include bacteremia, endocarditis, and orbital cellulitis2,3,4,5,6. Furthermore, they are emerging as causative agents of bloodstream infections in immunocompromised, neutropenic, and cancer patients that have undergone chemotherapy5,7,8,9.
The mechanisms underlying mitis group pathogenesis is obscure, because few virulence factors have been identified. The mitis group is known to produce H2O2, which has shown to play an important role in oral microbial communities10. More recently, several studies have highlighted a role for H2O2 as a cytotoxin that induces epithelial cell death11,12. S. pneumonia, which belongs to this group, has been shown to produce high levels of H2O2 that induces DNA damage and apoptosis in alveolar cells13. Using an acute pneumonia animal model, the same researchers demonstrated that production of H2O2 by the bacteria confers a virulence advantage. Studies on pneumococcal meningitis have also shown that pathogen-derived H2O2 acts synergistically with pneumolysin to trigger neuronal cell death14. These observations clearly establish that H2O2 produced by this group of bacteria is important for their pathogenicity.
Interestingly, it has also been shown that members of the mitis group S. mitis and S. oralis cause death of the nematode C. elegans via the production of H2O215,16. This free-living nematode has been used as a simple, genetically tractable model to study many biological processes. More recently, the worm has emerged as a model to study host-pathogen interactions17,18. In addition, several studies have highlighted the importance of studying oxidative stress using this organism19,20,21. Its short life cycle, ability to knockdown genes of interest by RNAi, and use of green fluorescent protein (GFP)-fused reporters to monitor gene expression are some of the attributes that make it an attractive model system. More importantly, the pathways that regulate oxidative stress and innate immunity in the worm are highly conserved with mammals20,22.
In this protocol, it is demonstrated how to use C. elegans to elucidate the pathogenicity caused by streptococcal-derived H2O2. A modified survival assay is shown, and members of the mitis group are able to kill the worms rapidly via the production of H2O2. Using members of the mitis group, a sustained biological source of reactive oxygen species (ROS) is provided, as opposed to chemical sources that induce oxidative stress in the worms. Furthermore, the bacteria are able to colonize the worms rapidly, which allows for H2O2 to be directly targeted to the intestinal cells (compared to other sources that have to cross several barriers). The assay is validated either 1) by determining the survival of the skn-1 mutant strain or 2) by knocking down skn-1 using RNAi in worms relative to the N2 wild-type and vector control treated worms. SKN-1 is an important transcription factor that regulates the oxidative stress response in C. elegans23,24,25. In addition to survival assays, a worm strain expressing a SKN-1B/C::GFP transgenic reporter is used to monitor activation of the oxidative stress response via the production of H2O2 by the mitis group.

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		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. oralis ATCC 35037</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. mitis ATCC 49456</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. gordonii DL1 Challis&#160;&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:381px;">E. coli OP50</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">E. coli HT115</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Worm Strains</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Strain</td>
			<td style="width:179px;">Genotype</td>
			<td style="width:119px;">Transgene</td>
			<td>Source</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">N2</td>
			<td style="width:179px;">C. elegans wild isolate</td>
			<td style="width:119px;"></td>
			<td>CGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">EU1</td>
			<td style="width:179px;">skn-1(zu67) IV/nT1 [unc-?(n754) let-?] (IV;V)</td>
			<td style="width:119px;"></td>
			<td>CGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">LD002</td>
			<td style="width:179px;">IdIs1</td>
			<td style="width:119px;">SKN-1B/C::GFP + rol-6(su1006)</td>
			<td>Keith Blackwell</td>
		</tr>
	</tbody>
</table>

studying oxidative stress caused by the mitis group streptococci in caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Members of the mitis group S. mitis, S. oralis, and S. gordonii rapidly killed the worms, as opposed to S. mutans, S. salivarius, and non-pathogenic E. coli OP50 (Figure 3A). The median survival for S. mitis, S. oralis, and S. gordonii was 300 min, 300 min, and 345 min, respectively. To determine if the killing was mediated by H2O2, catalase...

Watch this Scientific Journal Video about Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans at JoVE.com

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

studying-oxidative-stress-caused-mitis-group-streptococci

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JoVE Journal

Developmental Biology

7.1K Views.  University of Texas Health Science Center. The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Video: Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

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.

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.

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

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...