This protocol utilizes a regulatable promoter that allows controlled protein expression. In this experiment, we use a high copy two micron plasmid that expresses the N two domain from a methionine repressible promoter. Wild type yeast cells are transformed with this.
Plasmid and transformants are selected on plates lacking uol. After overnight growth. In selective media cells are synchronized by no cortisol mediated growth.Arrest.
Finally, and to expression is induced by transferring cells to media lacking methionine during the time course experiment. Protein concentration is monitored by western blotting and morphological phenotype development by microscopy. Hi, I'm Claudia in the Department of Biological Sciences, Purdue University.
I am de Bari Muji in the alar and I'M and also in the AL lab. Today we're gonna show you a procedure for analysis of development of a morphological phenotype as a function of protein concentration in body induct. We use this procedure in our laboratory to study the morphological and molecular aspects of a cell division phenotype observed upon overexpression of the N two domain of the endocytic adaptive protein absent two.
So let's get started. This protocol begins with a construction of a yeast strain that will express your protein of interest. In our case, we transformed the wild type yeast strain W 3 0 3 with high copy plasma DNA that contains nth two under the control of the methionine repressible promoter two millimolar methionine suppresses met 25 promoter activity.
While media lacking methionine allows maximal expression, pick six colonies from a plate containing recently transformed cells and inoculate in 50 milliliters of selective media with 0.2 millimolar methionine in a conical flask incubate overnight at 30 degrees Celsius with shaking at 200 RPM on the following morning. Estimate cell density by measuring the optical density of the culture at 600 nanometers. Transfer the equivalent of 20 OD 600 nanometers per milliliter of cells to a sterile centrifuge tube and harvest by centrifugation.
Re suspend the cells in YPD medium by vortexing to synchronize the cell cycle aole to a final concentration of 15 micrograms per milliliter from a stock of one milligram per milliliter in DMSO. Incubate for four hours at 30 degrees Celsius with shaking at 200 RPM. After four hours, check the percentage of cells arrested in mitosis.
By viewing under the microscope and counting the number of cells with equal sized buds proceed to the next step. Only if the arrest is greater than 90%Harvest the cells by centrifugation at 2200 RPM for five minutes. After three washes with ice cold water, resus, suspend the pellet in selective media lacking methionine at a cell density of about 0.5 OD 600 nanometers per milliliter.
Transfer half the volume of cells to a new sterile tube and add methionine to a final concentration of 0.2 millimolar. This will serve as a control for effects due to basal levels of protein expression. Return both cultures to the 30 degree Celsius incubator.
Cells are analyzed for phenotype development every hour for six hours at each time. Point transfer one milliliter of cell suspension of each culture to a sterile micro fuge tube and harvest the cells by centrifugation, aspirate the supernatant and re suspend the pellet in 70 microliters of media. Then add 30 microliters of methylene blue from a stock of one milligram per milliliter in sterile water.
Next, pipette about six microliters of this suspension onto a clean glass slide and place a cover slip over the drop. Press the cover slip gently to form a thin uniform layer of cells between the glass interfaces. Divide the cover slip into nine quadrants and take three images per quadrant.
The number of cells per field should be no larger than 30 cells to allow accurate quantification. Count the number of cells showing the phenotype under study, which in this example are budding yeast cells with elongated and or concatenated buds that fail to separate from each other. We also count the number of dead cells identified by their blue color since the cell division defect induced upon nth.
Two overexpression leads to cell death to visualize phenotype specific defects at every time. Point aliquot one milliliter of culture in a sterile micro fuge tube and harvest cells by centrifugation reus. Suspend the cell pellet in 100 microliters of one milligram per milliliter, calor white solution in sterile water and incubate for five minutes at room temperature in the dark.
Staining with calcior white enables visualization of the e cell wall. Wash the pellet three times with one XPBS. We suspend the final pellet in 100 microliters of PBS.
Observe the cells under the microscope and acquire images at 100 x magnification using UV optics for visualizing the cell wall. To visualize GFP tagged septin acquire images using a FS E filter, Quantify the percentage of cells with cell wall defects and septin mis localization using the method shown earlier by dividing the cover slip into nine quadrants and taking three images per quadrant for counting TimeLapse video. Microscopy of single yeast cells requires media embedded agros beds to make an agros bed.
First clean a glass slide with a concave depression with 70%alcohol and allow it to air dry while the slide is air drying. Boil 0.06 grams of agros in five milliliters of selective media lacking methionine in a microwave until the agros dissolves completely quickly. Pipette 200 microliters of the agros containing media in the slide.
Depression and invert another clean glass slide over it, making sure that no air bubbles are trapped underneath. When the gel solidifies, move the slide on top smoothly away from the depression slide, keeping their surfaces parallel at all times. This ensures that the surface of the agros bed is flush with the surface of the depression slide.
Clean the slide region surrounding the agros bed with a delicate tissue. The slide is now ready for use at time equaling four hours. Transfer one milliliter of cells from the culture to a sterile micro fuge tube and harvest by centrifugation.
Aspirate the supernatant and resuspend the cells in 100 microliters of fresh selective media lacking methionine. Apply petroleum jelly to the edges of the cover slip. Then place a drop of cell suspension at the center of the agros bed and spread uniformly by gently pressing a cover slip over it.
Seal the edges of the cover slip and fix its position by lining the edges with nail polish. Once the nail polish is dry, place the slide on a heated stage of a microscope. We use a Zeiss Axio 200 M microscope equipped with a Zeiss Axio camm, MRM monochrome digital camera to choose a field that contains no more than 10 cells spaced adequately because the field will get crowded over time.
As cells divide, take an image of the field every five minutes for about five hours. In order to avoid readjusting the focus repeatedly. We program the image acquisition software to automatically acquire a few Zack images at every time point.
The image with the best focus will be selected later for assembling the movie in order to ensure adequate heat for the cells, an external source of heat in addition to the heated stages provided by leaving the transmitted white light of the microscope on for the entire duration of imaging phenotype regression can be studied by assembling the images into a movie using Image J software. Here we show representative results of over expressing our protein of interest. Nth two.
First, the protein expression of nth two. During the time course of the experiment was determined lysates from cells expressing HA nth two were analyzed by western blotting using a monoclonal anti HA antibody as shown by the immuno blot cells grown in the presence of 0.2 millimolar methionine have minimal protein expression. However, cells grown in media lacking methionine show a consistent rise in concentration of S two over the time course of the experiment.
Next cells grown in the presence or absence of methionine for six hours were analyzed since a low nth two concentration is incapable of inducing a cell division phenotype or cell death only. A low percentage of cells are dead as indicated by their blue color after staining with methylene blue. Likewise, the number of nuclei per cell, cell wall and septin organization are normal as expected.
In contrast over expression of M two leads to a massive cell division defect and cell death as demonstrated by long chains of connected elongated buds that retain the blue color when stained with methylene blue. Most phenotypic cells are multinucleated as indicated by DPI staining. Calcior white staining demonstrates accumulations of the abnormal cell wall patches.
In addition, septin is grossly mis localized and disorganized. The phenotype quantification as a function of time is shown in this graph where the open circles represent zero millimolar methionine and the closed circles represent 0.2 millimolar methionine. We see here that as nth two concentration builds up in the cells over time, the percentage of cells showing the cell division phenotype also rises.
Control cells grown at 0.2. Milli methionine show that phenotype development is a function of elevated protein concentration and not because of cell culture conditions. Finally, phenotype development upon nth two overexpression is captured by time-lapse video microscopy.
After four hours of nth, two overexpression cells show a mild morphological cell division phenotype. As the movie progresses, we see abnormal and extended periods of apical bud growth, giving rise to very elongated buds. We also see events of new bud emergence before a cell separation event has taken place.
In addition, buds are observed to emerge from several regions of the mother's cell giving rise to a branch to appearance. We've just shown you how to characterize and analyze the development of a morphological phenotype as a function of protein concentration in budding list. By doing this procedure, it's important to remember to pet the cells at the recommended centrifugation speeds as faster speeds might damage the cells.
It's also important to remember to add methylene blue and chloride just prior to imaging as they are toxic to light east cells. So thanks for watching and good luck with your experiments.
