The overall goal of the following experiments in the genetically tractable metazoan CENO R elegance is to monitor protein misfolding at three levels, the organism, the cells, and the proteins. Behavioral phenotypes such as motility or thermo resistance, function as readouts of changes in protein homeostasis at the organismal level, temperature sensitive, or naturally occurring. Meta stable proteins are used to follow protein mis localization, which can be associated with protein misfolding.
An ex vivo partial digest assay monitors protein stability directly. Results can show changes in proteostasis capacity at different cultivation temperatures based on the thermo resistance assay, immunofluorescence microscopy, and western blood analysis. These procedures will be demonstrated by graduate students in my laboratory.
The behavioral assay will be demonstrated by ish ido and Shai will demonstrate the immuno staining and ANA flunking and sheron will demonstrate the partial digestion. This protocol for measuring temperature tolerance is accompanied by preparatory steps for synchronizing the animals and a movement assay. Both are described in the text protocol begin by collecting replicates containing at least 20 synchronized animals.
In 24, well-played wells containing 450 microliters of heat shock buffer. Supplement each well with nine microliters of cytoxin. Now transfer the plate to a heated bath.
The heat shock, temperature and duration strongly depend on the growth conditions. In particular, the cultivation temperature score the animal's survival by monitoring their die uptake. Using a fluorescent stereoscope with a TXR filter, the animals that take up the die are dead.
The main advantage of this technique of existing methods like kine, the tolerance by manual scoring is the use of visual readout of animals mortality, which makes this essay much faster and more reproducible. At the required stage, transfer at least 30 animals into a micro centrifuge tube containing M nine buffer. Wash the animals a few times with M nine by performing short, low speed centrifugation steps and Resus suspending them in M nine.
Then place the tubes on ice for a few minutes to cool down. Once cooled, remove the excess liquid and incubate the tubes. In 500 microliters of ice cold, 4%PARAFORM aldehyde.
The incubation time needs to be calibrated for each protein examined and should be around five to 30 minutes at room temperature. Now, wash the fixed animals three times with PBS tween at pH 7.2 by centrifugation. Centrifugations are always performed at 3000 RPM for one or two minutes unless specified otherwise.
In the hood, remove most of the supernatant and resuspend the pellet in one milliliter of beam mercaptoethanol solution. Then incubate the animals at 37 degrees Celsius overnight with gentle rocking the next day in the hood. Wash the animals three times with PBST by centrifugation and Resus.
Finish the wash with 50 to 100 microliters of PBST and remove most of this PBST after another centrifugation. Next, add 100 to 150 microliters of collagenase solution and incubate the animals at 37 degrees Celsius with strong agitation. Use a thermo mixer or a shaking incubator.
After five minutes, start making periodic checks under a stereo microscope. When one fifth of the animals are broken, stop the reaction by placing the tube on ice. This step is highly sensitive.
The duration of the treatment depends on the fixation conditions, and can change widely between strains. A prolonged incubation could result in the complete degradation of the animals. The conditions must be calibrated before each enzyme batch and the degradation should be monitored carefully.
Next, wash the animals twice in PBST. Collect the animals by centrifugation and remove most of the PBST. Then add one milliliter of a BA solution followed by incubation with gentle rocking for an hour at room temperature, collect the animals by centrifugation and resuspend them in 200 microliters of a b, A.Now add the primary antibody and begin an overnight incubation with gentle rocking at room temperature.
These parameters do need to be calibrated. Next, wash the animals by centrifugation. Resuspend them in a milliliter of a b, A and incubate them with gentle mixing for 15 minutes to an hour At room temperature.
At this point, an optional addition of fluorescent dye can be added. Next, repeat the a BA buffer wash three more times and finish the washes by Resus suspending in 100 microliters of a BA with the appropriate fluorescently tagged secondary antibody At a one to 100 dilution. Incubate the samples with rocking for several hours at room temperature protected from light to clear the secondary.
Perform three washes with a, b, a. Then mount animals on slides by first Resus, suspending them in 10 microliters of a b, a, and then using a wide bore pipette to transfer one or two microliters of the sample to each slide, add an equal volume of mounting medium and cover the slide. Gently seal the slide with nail polish.
The slides can be stored at negative 20 degrees Celsius for prolonged periods. Generally, individuals new to this protocol will struggle because the protocol is long and includes many steps that can affect its efficiency making troubleshooting difficult. Begin by collecting the animals from about 1560 millimeter plates into one or two milliliters of M nine buffer.
The animals should not be overcrowded. Wash the animals a few times with M nine buffer by centrifuging and resus suspending the animals. After the final spin, resus suspend the animals in 100 microliters of M nine buffer.
Then flash freeze the animals and using a chilled pestle and drill, grind up the sample while on ice. Frequently paused to check if animals are sufficiently ground. Once the animals are fully crushed, estimate the volume of the worm extract.
Then add an equal volume of two x worm lysis solution and incubate the sample on ice for 15 minutes. Next, remove the debris by a cooled centrifugation cycle and transfer the supernatant to a clean micro fuge tube. After a Bradford assay dilute the sample to three to 3.5 milligrams of protein per milliliter.
Then add freshly prepared chilled camo trypsin solution at a one to 1200 ratio. At this point, set the sample to incubate for an hour. During the incubation, periodically take 20 microliter aliquots and immediately mix them with SDS buffer, followed by five minutes at 96 degrees Celsius.
Run out the samples and follow with a Western blood analysis to determine the relative stability of the protein. While attempting this procedure, it's important to remember not to add protease inhibitor cocktail to the digest sample and instead we'll quickly and keep the samples on ice. The E 781 K ts mutation in the muscle chaperone on 45 strongly affects myosin folding and assembly and therefore affects animal motility.
When these mutants were grown at temperatures above 20 degrees Celsius, they were completely paralyzed, but not at 15 degrees Celsius. When wild type animals raised at 25 degrees Celsius were subjected to a prolonged heat shock, most survived. While only 30%survived if raised at 15 to 20 degrees Celsius to monitor the effective modifiers on the heat shock response, the control stress conditions were elevated to a 70%survival rate for wild type animals.
Animals were exposed to a 37 degree Celsius heat shock for six hours. On the first day of adulthood, survival was assayed by the cyt orange uptake assay. Dead animals are indicated by white arrows and live animals by yellow arrows.
The localization of several meta stable proteins were visualized using specific antibodies. For example, paramin ts mis localized to polycrystalline assemblies instead of to muscle sarcomere under restrictive conditions. Likewise, myosin TS forms disorganized filaments instead of the highly ordered filamentous structures at the restrictive temperature.
Animals expressing various TS mutant proteins co-expressed with myo three. GFP showed a rapid deterioration in myo filament organization as detected by monitoring GFP changes in myosin confirmation can reflect changes in the folding capacity of muscle cells. The proteolytic fragmentation pattern of myosin heavy chain, a co-expressed with UNC 45 Ts shows distinct characteristics compared with the digestion profile of myosin from wild type animals supporting the claim that myosin folding is affected by UNC 45.
Loss of function. Though this method can provide insight into the regulation of proteostasis in sea elegance, it can also be applied in other systems such as different model organisms.
