Name: measurements of physiological stress responses in c. elegans
Uploaded: 2020-05-21T13:30:00
Description: Watch this Scientific Journal Video about Measurements of Physiological Stress Responses in C. Elegans at JoVE.com

R.BZ. is supported by the EMBO long term fellowship and The Larry L. Hillblom Foundation. R.H.S is supported by grant 5F32AG032023-02 through the National Institute of Aging (NIA) and the Glenn Foundation for Medical Research Postdoctoral Fellowship. A.F. is supported by grant F32AG051355 through the NIA. H.K.G. is supported by grant DGE1752814 through the National Science Foundation Graduate Research Fellowship Program. M.G.M. is supported by 1F31AG060660-01 through NIA. A.D. is supported by the Thomas and Stacey Siebel Foundation, the Howard Hughes Medical Institute, and 4R01AG042679-04 and 5R01AG055891-02 from NIA, and 5R01ES021667-09 from NIEHS. We thank Larry Joe, Melissa Sanchez, Naame Kelet, and Anel Esquivel for significant technical assistance. We thank the Morimoto lab and the CGC (funded by NIH Office of Research Infrastructure Program P40 OD010440) for strains.

1. Standard growth conditions of temperatures &#38; OP50 vs HT115
<ol>
	<li>Standard growth and expansion
	<ol>
		<li>Grow a culture of OP50 in LB (Table 1) or equivalent media of choice for 24-48 h at ambient temperature (~22-25 &#176;C). Grow bacteria at room temperature as OP50 is an uracil auxotroph and there is a higher incidence of revertants (e.g., suppressor mutants) when grown at 37 &#176;C. Long-term storage of OP50 cultures is not recommended (max 1 week at 4 &#176;C).</li>
		<li>Seed a volu...

Here, methods to interrogate cellular stress responses in C. elegans, using fluorescent transcriptional reporters and physiological stress survival assays are described. The reporters all utilize GFP expression driven under the promoter of a downstream transcriptional target of the transcription factors involved in mounting cellular stress responses. The use of hsp-4p::GFP modulated by XBP-1s-mediated UPRER, hsp-6p::GFP controlled by ATFS-1-mediated UPRMT, gst-4p::GFP<...

The ability of an organism to respond to changes in the intra- and extracellular environment is crucial for its survival and adaptation. This is accomplished on a cellular level through numerous protective pathways that ensure the integrity of the cell. While numerous cellular components are subject to stress-associated damage, one major involvement of cellular stress responses is to repair and protect the homeostasis of the cellular proteome. However, the compartmentalization of proteins into special structures, called organelles, poses a challenge for the cell, as it cannot rely on one centralized form of protein quality control to ensure that all the proteins within the cell are properly folded and functional. Therefore, to deal with perturbations to their proteins, organelles have evolved dedicated quality control mechanisms, which can sense misfolded proteins and activate a stress response in an attempt to alleviate the stress within that compartment. For example, the cytosol relies on the heat shock response (HSR), while the endoplasmic reticulum (ER) and mitochondria rely on their compartment-specific unfolded protein responses (UPR). The OxSR serves to alleviate the toxic effects of reactive oxygen species (ROS). Each stress response is triggered in the presence of cellular challenges and environmental insults and induces a tailored transcriptional response. The hallmarks of these responses include synthesizing molecules that re-fold misfolded proteins (such as chaperones) targeted to the proper organelle, or alternatively, remove damaged proteins by protein degradation. Failure to activate these stress responses results in accumulation of damaged proteins, cellular dysfunction propagated to systemic failure of tissues, and eventually death of the organism. The function and regulation of the different stress responses are reviewed elsewhere1.
Many insights regarding the regulation and activity of cellular stress responses have been attributed to the nematode, Caenorhabditis elegans, a multicellular model organism in genetic research. Nematodes not only allow studying the activation of stress responses on the cellular level, but also on the organismal level; nematodes have been used to study the effects of genetic perturbations or exposure to drugs and pollutants on their growth and survival. Their quick generation time, isogeny, transparency, genetic tractability, and ease of use during experimentation make them ideal for such studies. Additionally, the relatively quick physiological response to stress (between hours and a few days) and the evolutionary conservation of cellular pathways make nematodes a prominent tool in studying stress resistance.
There are two commonly used E. coli strains used as a food source to grow C. elegans: standard OP50, a B strain in which most experimentation has been historically performed2 and HT115, a K-12 strain that is used for almost all RNAi experiments3,4. It is important to note that there are significant differences between OP50 and HT115 bacterial diets. Growth on these different bacterial sources has been shown to cause major differences in metabolic profile, mitochondrial DNA copy number, and several major phenotypes, including lifespan5. Some of these differences are attributed to Vitamin B12 deficiency associated with growth on OP50 bacteria, which can result in defects in mitochondrial homeostasis and increased sensitivity to pathogens and stresses. All of these phenotypes have been shown to be alleviated by growth on HT115 bacteria, which have higher levels of Vitamin B126. Therefore, it is recommended that all experiments on physiological stress responses be performed on HT115 bacteria, regardless of the necessity of RNAi conditions. However, due to the ease of maintaining animals on OP50, all standard growth (i.e., maintenance and amplification of animals) can be performed on OP50, as significant differences in the experimental paradigms described here were not detected in worms maintained on OP50 as long as they were moved to HT115 post synchronization (i.e., from hatch post-bleaching with or without L1 arresting) until experimentation.
Here, the characterization of the activity of cellular stress responses using two functional methods is described. It should be noted that the protocols presented are primarily focused on cellular stress responses and their impact on protein homeostasis. First, fluorescent transcriptional reporters are utilized, which are regulated by endogenous gene promoters that are specifically activated in response to different cellular stresses. These fluorescent transcriptional reporters are based on the transcriptional induction of specific genes that are natively part of the stress response. For example, HSP-4, a heat shock protein orthologous to the human chaperone HSPA5/BiP, is activated upon ER-stress and localizes to the ER to alleviate the stress. In conditions of ER stress (e.g., exposure to tunicamycin), a green fluorescent protein (GFP), placed under the regulation of the hsp-4 promoter, is synthesized in high levels as can be assessed by fluorescent microscopy or quantitatively measured using large-particle flow cytometry of nematodes7. Similarly, the promoter of a mitochondrial chaperone, hsp-6 (orthologous to mammalian HSPA9), is utilized to monitor the activation of the UPRMT8, and the promoter of the cytosolic chaperone hsp-16.2 (orthologous to the human crystallin alpha genes) is used for assessing the activity of the HSR9. These reporters allow a rapid characterization of the pathways activated in response to various perturbations.
Often, the reporters presented here are imaged using microscopy, which provides a qualitative output of the activation of stress responses. However, while imaging techniques provide both information on intensity and tissue location of the reporters described above, its quantification is not always accurate or robust. While it is possible to quantify fluorescent activation using imaging analysis tools, these methods are relatively low throughput and sample size is small, due to the relatively low number of animals imaged. The ease and ability to obtain large quantities of animals quickly make C. elegans an ideal model system to assay the activation of fluorescent stress reporters through the use of a large particle flow cytometer. A large-particle flow cytometer is capable of recording, analyzing, and sorting based on size and fluorescence from many live animals. Using this method, it is possible to get the fluorescent intensity, size, and also spatial (2D) information for thousands of worms. The system is controlled using FlowPilot, which allows for real-time data acquisition and analysis of the measured parameters. Here, methods for both microscopic imaging and quantitative analysis using a large-particle flow cytometer are offered as methods to measure the activation of stress responses.
Beyond reporter analysis, the sensitivity or resistance of animals to stress can be measured using physiological stress assays. This is achieved by exposing animals to stressful environments that activate specific cellular stress pathways. Here, several methods are provided to measure sensitivity of whole animals to specific types of stressors.
ER stress is applied to C. elegans by using the chemical agent, tunicamycin, which blocks N-linked glycosylation, causing accumulation of misfolded proteins in the ER10. In C. elegans, growth upon exposure to tunicamycin results in major perturbations in ER function, and a significantly decreased lifespan11. By measuring the survival of animals on tunicamycin-containing plates, ER stress sensitivity of animals can be quantified. For example, animals with ectopic UPRER induction and thus increased resistance to protein misfolding stress in the ER have an increased survival upon tunicamycin exposure compared to wild-type animals12.
Oxidative and mitochondrial stress is applied to C. elegans by exposing animals to the chemical agent, paraquat. Paraquat is a commonly used herbicide, which causes superoxide formation specifically in the mitochondria13. Due to the specific localization of mitochondria-derived reactive oxygen species (ROS), paraquat assays are often used as a &#34;mitochondrial&#34; stress assay. However, superoxide is rapidly converted into hydrogen peroxide by mitochondrial superoxide dismutases (SODs)14. Hydrogen peroxide can subsequently diffuse out of the mitochondria and cause oxidative stress in other compartments of the cell. Therefore, we describe paraquat survival assays as measuring sensitivity to both mitochondrial and oxidative stress (other oxidative stress assays can be found15).
Thermotolerance assays are performed in C. elegans by placing animals in elevated temperatures. Ambient temperatures for nematodes are ~15-20 &#176;C and thermal stress is induced at temperatures above 25 &#176;C16,17. Thermotolerance assays are generally performed at temperatures ranging from 30-37 &#176;C, as animals exhibit major cellular defects at this temperature, and survival assays are completed within 24 hours16,18. Here, two alternative methods are provided for performing thermotolerance assays: growth at 34 &#176;C and growth at 37 &#176;C. Together, the protocols presented here can be utilized to perform large-scale screens when combined with standard gene knock-down using RNA interference or chemical drug libraries.
The protocol can be broken into 4 broad procedures- growth of C. elegans and preparation for imaging (sections 1 and 2), imaging of transcriptional reporters using fluorescent microscopy (sections 3-5), quantitative measurements of reporters using a large-particle flow cytometer (section 6), and physiological assays to measure stress sensitivity in C. elegans (section 7).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td>for mitochondrial stress</td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>Fisher Scientific</td>
			<td>DF0118072</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>BD Difco granulated agar</td>
			<td>VWR</td>
			<td>90000-782</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>VWR</td>
			<td>97061-904</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>BioPioneer</td>
			<td>C0051-25</td>
			<td>for RNAi</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>57-88-5</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>COPAS Biosorter</td>
			<td>Union Biometrica</td>
			<td>350-5000-000</td>
			<td>equipped with a 488 nm light source.</td>
		</tr>
		<tr>
			<td>COPAS Cleaning Solution</td>
			<td>Union Biometrica</td>
			<td>300-5072-000</td>
			<td>to use with COPAS</td>
		</tr>
		<tr>
			<td>COPAS Sheath Solution</td>
			<td>Union Biometrica</td>
			<td>300-5070-100</td>
			<td>to use with COPAS</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td>solvent for drugs</td>
		</tr>
		<tr>
			<td>IPTG dioxane free</td>
			<td>Denville Scientific</td>
			<td>CI8280-4</td>
			<td>for RNAi</td>
		</tr>
		<tr>
			<td>LB Broth Miller</td>
			<td>Fisher Scientific</td>
			<td>BP1426500</td>
			<td>for LB</td>
		</tr>
		<tr>
			<td>M205FA stereoscope</td>
			<td>Leica</td>
			<td>10450040</td>
			<td>equipped with a Leica DFC3000G monochromatic CCD camera, standard Leica GFP filter (ex 395-455, EM 480 LP), and LAS X software</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>VWR</td>
			<td>EM-MX0070-3</td>
			<td>for NGM plates, M9</td>
		</tr>
		<tr>
			<td>Paraquat</td>
			<td>Sigma-Aldrich</td>
			<td>36541</td>
			<td>for oxidative/mitochondrial stress</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>for bleach soluton</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>VWR</td>
			<td>EM-PX1570-2</td>
			<td>for NGM plates</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>VWR</td>
			<td>EM-PX1565-5</td>
			<td>for M9</td>
		</tr>
		<tr>
			<td>Revolve</td>
			<td>ECHO</td>
			<td>75990-514</td>
			<td>equipped with an Olympus 4x Plan Fluorite NA 0.13 objective lens, standard Olympus FITC filter (ex 470/40; em 525/50; DM 560), and an iPad Pro for camera and to drive ECHO software</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>71289-50G</td>
			<td>for imaging</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>EMD Millipore</td>
			<td>SX0420-5</td>
			<td>for NGM plates, M9</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>VWR</td>
			<td>71003-472</td>
			<td>for M9</td>
		</tr>
		<tr>
			<td>Tert-butyl hydroperoxide</td>
			<td>Sigma-Aldrich</td>
			<td>458139</td>
			<td>for oxidative stress</td>
		</tr>
		<tr>
			<td>Tetracycline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T7660-5G</td>
			<td>for RNAi</td>
		</tr>
		<tr>
			<td>Tunicamycin</td>
			<td>Sigma-Aldrich</td>
			<td>T7765-50MG</td>
			<td>for ER stress</td>
		</tr>
	</tbody>
</table>

measurements of physiological stress responses in c. elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Using transcriptional reporters to measure activation of stress responses 
Here, fluorescent transcriptional reporters are used, which serve as robust tools to measure activation of most stress responses in C. elegans. GFP expression is driven under the promoter of canonical targets of master transcriptional regulators involved in responding to compartment-specific stresses. A comprehensive list of commonly used transcriptional reporters is available in <str...

Watch this Scientific Journal Video about Measurements of Physiological Stress Responses in C. Elegans at JoVE.com

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

These methods can be used to characterize stress responses in C.elegans and to determine whether genetic or pharmacological perturbations affect the activation of protective cellular pathways. These methods allow rapid and high-throughput testing of hundreds of perturbations such as gene knockdown both on the molecular and physiological levels. To ensure the assay is performed properly, include positive and negative controls.Synchronize healthy, well-fed animals to the proper stage and use fresh plates for the experiment. Visualization of micromanipulation of the worms with a pick can help new users understand how the worms can be lined up and assessed for viability. Demonstrating the procedures will be Phil Frankino, Holly Gildea, and Melissa Metcalf, graduate students in our laboratory.To use animals expressing GFP under the promoter of the heat shock protein four for activation of the endoplasmic reticulum unfolded protein response, grow synchronized reporter animals at 20 degrees Celsius until the L4 stage and wash the worms off the plate using M9 medium and transfer into a tube. After collecting the worms by centrifugation, replace the M9 with 25 nanograms per milliliter of the drug of interest in M9 or dimethyl sulfoxide in M9 in the control animal tubes. Then place the worms on a rotating platform for three to four hours at 20 degrees Celsius.At the end of the incubation, centrifuge the worms to settle before replacing the treatment solution with 15 milliliters of M9 alone. After two washes, transfer the animals to NGM plates or NGM RNA interference plates as appropriate and allow the worms to recover overnight at 20 degrees Celsius. When the worms have recovered, add five to 10 microliters of 100 millimolar sodium azide onto a standard NGM plate without bacteria and place a plate of worms under a dissecting microscope.Transfer 10 to 20 animals from the plate into the spot of sodium azide. The animals should seize movement shortly after landing in the salt solution. Once the sodium azide has evaporated, line up the animals in the desired imaging setup with the anterior and posterior sides in the same orientation for all of the animals and immediately place the plate of animals under a stereo microscope.Launch the stereo microscope imaging software and under the acquisitions tab, click open projects and folder to open a new project. Right-click and select rename to rename the folder. Position the worm sample under the microscope objective and use the Brightfield setting to locate the correct focal point of the worms to minimize fluorescent bleaching.The center of the sample will be the point at which the line of eggs is clearly visible and not fuzzy. Set the exposure time to ensure that pixels are not saturated and also to ensure that the signal is above the detection limit. After setting the exposure time, zoom, focus, and Brightfield condensers to the appropriate settings, click the capture image button to acquire an image.For imaging using a compound wide-field microscope, place the baseline control treatment plate onto the compound wide-field microscope stage and use the touch pad to launch the control program. Create a new album and filename and position the plate under the objective lens. Use the baseline control and positive control plates to set the exposure time and fluorescence intensity so that the signal is visible but not saturated.Then save Brightfield and GFP FITC images. To quantify the induction of the reporter by large particle flow cytometry, first turn on the cytometer lasers and open the cytometer software. Click start to turn on the lasers in the software window and click run to initiate the laser in the argon laser control popup window.When the laser reaches around 12 milliwatts and the 488 light source level goes up to around 12, click done and check the four pressure values. If the values look similar to those observed in the original setup, check the pressure OK box. Next, to make sure there are no air bubbles or debris blocking the flow of sheath and sample through the flow cell, click clean several times.Then switch off sort and on sheath to restart the flow. To check the flow rate, collect the sheath for 60 seconds. Collect the sheath into a 15 milliliter tube for 60 seconds.The flow rate should be nine to 10 milliliters per minute. To run the samples, adjust the laser photomultiplier power to a level high enough that the signal is above the detection limit but not higher than the saturation limit. Perform size gating to exclude bubbles, debris, eggs, and other unwanted small particles and to include only the animals of interest such as adults.When the screening parameters have been set, add the prepared worms to a cup and click acquire. Watch to make sure that all of the liquid is not taken up into the machine as this will cause the flow cytometer to take in air and create bubbles within the detector. When the sample is low and/or enough animals have been collected, click stop.To store gated data based only on the size constraints, click setup, data storage, only gated, and store gated to save the data. Then rinse the collection cup with deionized water and remove the wash by vacuum three times before repeating the analysis with the next sample. To measure the mitochondrial and oxidative stress sensitivity, add 50 to 75 microliters of Paraquat in M9 to eight to 10 wells per condition of a flat bottom 96-well plate and transfer eight to 10 worms per condition to each well of Paraquat.Every two hours, tap the plate gently to cause the live animals to thrash or bend to allow scoring of the number of dead animals per well. To measure the temperature sensitivity of the animals, incubate the worms for three to four days at 20 degrees Celsius before plating 10 to 15 animals per plate per condition for a total of four to six plates. Then place the plates into a 34 or 37 degree Celsius incubator and score the worm survival every two hours as just demonstrated.The hsp-4 reporter has minimal basal impression in the absence of stress but exhibits a robust GFP expression when the animals are exposed to endoplasmic reticulum stress using the drug Tunicamycin. These differences can also be quantified using a large particle flow cytometer. Moreover, the induction of the transgene under endoplasmic reticulum stress can be completely supressed by knockdown of XBP-1 via RNA interference.Similar assays can be performed to measure mitochondrial stress, oxidative stress, and heat stress. Whole animal physiological responses to stress can also be measured using several survival assays. For example, exposure to Tunicamycin is used to measure endoplasmic reticulum stress sensitivity.The knockdown of the XBP-1 gene results in a significant increase in sensitivity to Tunicamycin. Moreover, exposure to the chemical agent Paraquat is used to measure oxidative and mitochondrial stress sensitivity. Knockdown of the DAF-2 gene results in a significant increase in resistance to Paraquat.Thermotolerance can be measured by determining the survival of animals at elevated temperatures and can be plotted as a survival curve. These assays should be performed at least four to six times and all of the replicates should be plotted against each other as thermotolerance demonstrates an incredibly high variability as compared to other stress assays. When imaging animals, it is critical to set the parameters such that there is no over or under saturation of signal and to ensure that the same parameters are used throughout the experiment.The imaging protocols described here are amenable for large scale screening including genome-wide screens and are great for both exploratory and confirmatory studies which can then be followed up by the physiological assays described.

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