The overall goal of this procedure is to use yeast as an experimental platform to assess and analyze the toxicity associated with poly glutamine. Expansion proteins following growth of yeast under conditions that repress poly glutamine expression. Three, complimentary growth assays of yeast cells expressing a toxic poly glutamine Expansion protein are performed to reliably assess toxicity in the first growth assay demonstrated.
Here, poly glutamine toxicity is monitored through a plating assay. As a second complimentary growth assay, A spotting assay is utilized to detect even subtle differences in cell growth. The most quantitative approach is shown in the third assay where yeast cells are grown in liquid cultures and toxicity is monitored through observation of optical density At 600 nanometers, ultimately reproducible and quantitative results can be obtained.
That document poly glutamine toxicity in yeast cells through careful examination of their growth on plates and in liquid cultures. This method can answer key questions on the feed of protein misfolding disorders such as deciphering genetic and cellular underpinnings of the toxicity associated with misfolded proteins, including poly glutamine proteins. A systematic analysis has established the precise amino acid sequence of a poly Q expansion protein that is required to produce toxicity in yeast.
This toxic poly Q expansion protein contains an amino terminal flag tag followed by 17 amino acids from the original sequence of the Huntington protein, a poly Q region, and a carboxy terminal fusion to a fluorescent protein yeast cells expressing the otherwise toxic poly Q expansion proteins may not show any growth defect due to spontaneous suppressors of poly Q toxicity, which can jeopardize the successful outcome of any experiment that aims to characterize poly Q toxicity or its modifiers. In order to avoid the frequent occurrence of these spontaneous suppressors, be sure to take precautionary measures, including the use of fresh yeast cells for toxicity experiments. Avoid storing yeast for extended periods of time and frequently retrieve fresh yeast colonies from frozen stocks.
Regularly monitor the expression and aggregation of toxic poly Q expansion proteins by fluorescent microscopy. Keep the yeast cells in media that represses the expression of the toxic proteins at all times. Before starting any toxicity measurements.
As a final preventive measure, use multiple independent transformants for each experiment. To begin, the growth assays demonstrated in this protocol obtain yeast cells that encode for expression of the poly Q expansion proteins using three milliliters of selective yeast media with glucose. As the sole carbon source inoculate three cultures from three independent transformants incubate the cultures overnight at 30 degrees Celsius.
Under these conditions, the cells do not express the poly Q expansion protein due to repression by glucose. Avoid letting the overnight cultures overgrow by keeping the optical density at 600 nanometers below one to monitor defects through growth on plates. First, perform a one to 1000 dilution of an overnight yeast culture with an OD 600 of 0.5 with selective media containing glucose evenly spread 50 microliters of each diluted culture on one plate with selective medium containing glucose as the sole carbon source to result in approximately 700 colonies per plate for each diluted culture.
Also plate onto selective medium containing galactose. As the sole carbon source, incubate the plates for three to four days at 30 degrees Celsius after the incubation. Take photographs of each plate and count the number of colonies on the glucose and galactose plates.
Under ideal conditions, there should be no or very few colonies in the galactose plate when using yeast cells expressing a highly toxic poly Q expansion protein. The spotting assay is more quantitative than the plating assay and can thus reveal even subtle differences in poly Q toxicity. Within the same experiment on the same plate, to begin spotting assays in medium with glucose dilute the overnight cultures grown to an OD 600 of 0.1 pipa 200 microliters of each diluted culture into sterile 96 well plates and prepare five five-fold serial dilutions in sterile water using a multichannel pipette using a frogger transfer.
The cell suspensions onto plates containing selective media with glucose as the sole carbon source. In addition, transfer the cells onto plates containing selective media with galactose as the sole carbon source after allowing the plates to dry incubate them at 30 degrees Celsius for three to four days before taking photographs of each plate. The liquid growth assay is the most quantitative of the three assays described here to monitor poly Q toxicity in yeast.
The bio screen C is an instrument that automatically measures the optical density of yeast cultures in 100 well plates while incubated at defined temperatures with defined agitation. Other methods for growing yeast cells and measuring their optical density can also be used to begin spin down the yeast cells that were grown overnight in minimal media containing glucose as the sole carbon source. Following centrifugation, wash the cells in three milliliters of sterile water.
Repeat the wash twice, spinning the cells down between each wash using medium with galactose. Dilute the washed cells to an OD 600 of 0.1. Then fill each well of the 100 well bio screen C plate with 300 microliters of the diluted yeast cultures and insert the plate into the instrument.
Open the easy bio screen experiment program. Determine the number of samples to monitor. Set the temperature to 30 degrees Celsius.
Set the length of the experiments to three days and set the measurement intervals to 15 minutes. Also, set the filter to 600 nanometers brown and set the shaking mode to 15 seconds before each measurement at medium strength. After setting the parameters, begin the experiment.
The bio screen, see instrument, and the attached software will produce Excel spreadsheets of each data point taken during the experiment. Using Excel growth curves can be prepared for each sample and the growth of different samples can be compared. Shown here are results from the plating assay.
Approximately 700 yeast cells were spread on plates and incubated for three days at 30 degrees Celsius on the plate containing glucose medium. The expression of the toxic poly Q expansion protein is not induced. Conversely, when the yeast cells were plated on galactose medium, the expression of the toxic poly Q expansion protein is induced.
Note that in the experiment shown here, no spontaneous suppression occurred. Similarly, in the spotting assay expression of the toxic Poly Q expansion protein is observed on galactose and not glucose. Medium five, serial fivefold dilution of yeast cells harboring either a non-toxic poly Q protein or a toxic poly Q expansion protein or spotted on glucose plates that repress the expression of the proteins or on galactose plates that induce their expression in the bio screen C experiment.
The growth of yeast cell cultures expressing either a non-toxic poly Q protein or a toxic poly Q expansion protein were monitored by the bio screen C instrument as described in this video, the effect of expression of the toxic poly Q expansion protein on cell growth is evident here. After watching this video, you should have a good understanding of how to monitor the toxicity of poly glutamine proteins in yeast. The liquid growth assay is the most quantitative of the three assays, as it can even detect subtle differences in poly glutamine toxicity.
But please remember to combine it with at least one of the two plating assays to avoid problems with spontaneous suppresses of poly glutamine toxicity.
