Download Signal Transduction

Signal Transduction
CELL BIOLOGY LAB 10
Before Coming to Lab:
• Read the entire lab carefully!
• Would you expect the α factor receptor to be on the plasma membrane or in the cytosol?
Please explain your reasoning. What technique could you use to find out if your prediction
is correct?
• Assume that the NCC1 gene in yeast encodes a protein that is necessary for α factor signal
transduction. Further assume that wildtype cells not treated with α factor produce 1 unit of
β-galactosidase activity and that wildtype cells that were treated with α factor produce 10
units of β-galactosidase activity. How much β-galactosidase activity do you expect in cells
missing the entire ncc1 gene if those cells were not treated with α factor? How much
activity would you expect if the cells were treated with α factor? Under each of these two
conditions, would the cells be shmooing? Record your answers in your lab notebook.
• Assume that the NCC2 gene in yeast encodes a protein that acts as a negative regulator of
this pathway. How much β-galactosidase activity do you expect in cells (with and without α
factor) missing the entire ncc2 gene? Would these cells shmoo with or without the α factor?
Record your answers in your lab notebook.
Background:
In class, we’ve examined several signal transduction pathways in terms of what proteins are
involved, what they each do and their second messengers. How did researchers determine that the Ras
protein functions in the EGF pathway? How did we determine that cAMP is not important for EGF
signal transduction? Obviously, there were a long series of meticulous experiments that examined
which proteins and compounds function together. This week, we are going to conduct a similar
research project to identify some proteins and second messengers that are necessary for one specific
signal transduction pathway.
Often, studying these pathways is very difficult. This is because working with human cells or
vertebrate organisms takes a long time and its difficult to add or remove genes. Therefore, we will
work in with a model organism for today’s lab. Our organism of choice this week is everyone’s
favorite ‘lower’ eukaryote, Saccharomyces cerevisiae, or yeast. Yeast exists in two different ‘genders’
that are properly referred to as mating types. Rather than calling them ‘male’ and ‘female’ cells, we
refer to the two cell types as a and α cells. If a bunch of a cells are growing in a test tube they can
keep dividing mitotically as long as nutritents are present. But if an a cell detects that an α cell is
nearby, it will stop going through mitosis (in other words, stop the cell cycle) and will prepare to mate
with the α cell. How do a cells detect the presence of a nearby α cell? The α cells secrete a 13-amino
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acid peptide called α-factor which diffuses from the α cell to the a cell. So in our model yeast system,
the α factor essentially acts as a hormone, communicating information from one cell to another.
As you might expect, preparing to mate involves many different activities and genes; we are
going to look at two specific responses to α-factor. One response is that the a cell will radically
reorganize it’s actin cytoskeleton to grow a “germ tube” towards the source of the α factor. Yeast cells
are nonmotile but they need some way to get to the α cell with which they will fuse. Cells making
germ tubes are also referred to as “shmoos” since they look like a character from Al Capp’s comic strip
“Li’l Abner” that was popular in the 1950’s. Some shmooing cells are shown below. Compare these
images with your drawings of mitotically dividing yeast cells from Week 2.
Eight examples of “shmoos”
Additionally, the preparation for mating includes activing the expression of several matingspecific genes whose expression is normally repressed. One of these genes is called FUS1 and is
required to allow the fusion of the nuclei. Cells that are capable of responding to α-factor should
strongly induce the transcription of FUS1.
At this point, we’ve got both ends of the signal transduction pathway. At the front end, we
know the hormone, α factor. At the back end, we have two distinct results: shmooing and activation of
the FUS1 gene. What’s in between? That’s the signal transduction pathway that we’re attempting to
discover.
We are going to start by making some guesses about what proteins might be involved in the α
factor signal transduction pathway. There are eight different proteins that might be involved in our
pathway.
They
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are…
Name
1 Ste2
2 Far1
3 Gpa2
4 Ras2G19V
5 Pde2
6 Mpk1
7 Tor1
8 Hog1
Description
Ste2 is a transmembrane protein that is known to interact with certain
heterotrimeric G proteins. In the mutant, this gene has been deleted.
Activated Far1 binds to a cyclin-CDK complex and inhibits its function to
arrest the cell in the cell cycle. The gene is deleted in the mutants that we have.
Gpa2 is the Gα subunit of a heterotrimeric G protein found in these yeast and is
deleted in these mutant cells.
Ras2 is the yeast version of the human Ras protein that we talked about in
class. This mutant version of Ras is locked in a permanently activated (i.e.,
GTP-bound) state.
Pde2 is a phosphodiesterase that breaks down cAMP. The gene is deleted in
these mutant cells.
Mpk1 is a kinase that phosphorylates proteins only when the cell wall has been
altered. In these mutants, the Mpk1 gene is deleted.
Tor1 is a cytosolic kinase whose activity is regulated by the nutritional state of
the cell. These mutants have a permanently activated Tor1 kinase.
Hog1 is a protein kinase that is located in the cytoplasm. Stressed cells are
known to activate the Hog1 kinase. In this mutant, the gene is deleted.
Take a moment to think about these different proteins. Notice that most of these mutations
would be expected to completely block the function of the protein. These are often referred to as a
loss-of-function mutation. In a few cases, the mutation causes a protein to be always active, a situation
called a gain-of-function mutation. Furthermore, some of these mutated proteins would be expected to
activate the pathway and some would be expected to deactivate the pathway. One of these proteins
directly affects a second messenger within the cell. Recall that second messengers are small molecules
(i.e., not proteins) that made in large quantities as an intracellular message.
How will we measure if the signal transduction pathway has been activated?
Assaying
shmooing is fairly easy. We will simply look at the cells under the microscope and determine what
percentage of the cells have started to from a germ tube. For our other outcome, we could determine
the amount of FUS1 mRNA that is made by a northern blot, but that’s a difficult experiment. We
could also measure the amount of Fus1 protein that is made using a western blot, but that is also fairly
difficult and time-consuming. We’re going to use a simpler method to find out when the pathway is
‘on’. We expect that an activated pathway will lead to a more active FUS1 promoter but it shouldn’t
really matter what gene lies beyond the promoter. It’s really the activity of the FUS1 promoter that
we’ll measure in today’s experiment.
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All of the yeast cells we will use this week contain a circular piece of DNA known as a
plasmid. The specific plasmid we have is called pBH315 and is diagrammed below. In pBH315, the
FUS1 promoter has been artificially placed upstream of the lacZ gene. The lacZ gene normally isn’t
found in eukaryotes like yeast at all! This is a gene from the bacteria E. coli but (because the genetic
code is universal) a lacZ mRNA can
be translated in yeast. The protein
encoded by the lacZ gene is called
β-galactosidase and is normally
used by the bacteria to digest the
carbohydrate lactose.
purposes,
the
For our
β-galactosidase
protein is simply an enzyme that is
very easy to assay. In our yeasts, if
α factor is present, it should activate
its signal transduction pathway
which will lead to the FUS1
promoter being activated.
An
activated FUS1 promoter will cause the lacZ gene to be transcribed, resulting in β-galactosidase
protein being produced. It’s the β-galactosidase that we can actually measure to determine if the
pathway has been activated or not. lacZ is serving as a reporter gene for the FUS1 promoter as it
‘reports’ how active the promoter has been.
We will measure the activity of the β-galactosidase using a simple
enzyme assay. We will add o-nitrophenylgalactopyranoside (ONPG, shown
on the left) as the substrate for the enzyme. ONPG is a colorless compound
that can be cleaved by β-galactosidase to release o-nitrophenol and galactose.
O-nitrophenol is yellow (λmax of 420 nm) so it’s concentration can be easily
measured with the spectrophotometer.
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Lab Procedure:
Each pair of students will determine how one particular change in the yeast affects the amount of βgalactosidase activity. We’ll pool our data from both lab sections so we can analyze all seven of the
mutants. Some of these mutants will be analyzed by more than one group. Overall, we will have
collectively generated an impressively large data set that will allow us to draw some conclusions about
the α factor signal transduction pathway in Saccharomyces cerevisiae.
Each group will look at four test tubes of yeast.
Tube A: wildtype cells without α factor.
Tube B: wildtype cells with α factor.
Tube C: mutant cells without α factor.
Tube D: mutant cells with α factor.
Realize that tubes A and B are identical for every group and serve as controls. Each group will
perform four β-galactosidase assays. For example, Group 5 will pick up the tubes labelled 5A, 5B, 5C,
and 5D to determine the amount of β-galactosidase enzyme in those cells.
Lysing your Yeast Cells
1. Pipet 1.5 mL of your yeast culture into a labelled microfuge tube. (Don’t throw out the rest of your
cultures; you’ll need them for looking at shmoos). Pellet your yeast cells by centrifuging for about
15 seconds. Don’t forget to balance the microcentrifuge before starting it!
2. Add 300 μL of the Lysis Buffer to each tube and resuspend the yeast cells by gently pipetting up
and down. Leave your tubes on ice when you’re not working with them.
The Lysis Buffer contains SDS which is a strong detergent to help break up the cell
membrane. It also contains the compound β-mercaptoethanol which will reduce disulfide
bridges in the yeast cell wall to help break that apart. The β-mercaptoethanol also makes the
Lysis Buffer very smelly so try not to spill it.
3. Using a small spatula, carefully add about 100 μL of glass beads to each tube.
4. Cap the tubes and vortex them vigorously for one minute. Return the tubes to the ice (to let them
cool down) for one minute. Repeat the vortexing/icing until you’ve vortexed all your cells four
times.
5. Centrifuge the tubes for one minute.
6. Remove the supernatant and discard the pellet with the glass beads. The supernatant contains
water-soluble proteins released from lysed yeast cells.
7. Pipet the supernatants into four new clean microfuge tubes that are labelled. This solution is your
lysate. Keep it on ice as much as possible to limit denaturation.
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Determining β-Galactosidase Activity
1. Pipet 100 μL of each lysate to a new labelled microfuge tube. In a another tube, put 100 μL of
Lysis Buffer (this will serve as your blank at the end).
2. Add 800 μL of Z Buffer to each tube. The Z Buffer provides a good pH and salt concentration of
the enzyme to work efficiently. Add 100 μL of ONPG to each tube.
3. Let the enzyme and the substrate incubate together at room temperature for thirty minutes.
4. Stop the reaction by adding 500 μL of 1M Na2CO3. The sodium carbonate increases the pH which
denatures the enzyme so the reaction ends.
5. Measure the absorbance at 420nm for each sample.
Determining the Total Protein Concentration in your Lysate
It’s not hard to imagine that one of your four tubes might have had a few more yeast in it or
that one group might have been more efficiently lysed than another. To control for that possibility, run
a Bradford Assay on each of your four lysates (you may re-use a standard curve from a previous lab) to
determine the protein concentration in each lysate. With this information, you can report the specific
activity of β-galactosidase per milligram (mg) of total protein.
Measuring Shmoo Frequency
When you look at cells under a microscope for shmoos, there will be some tough judgment
calls. Is that a small bud or the beginning of a shmoo? If you know that the cells you are looking at
are “supposed” to be shmooing, it is easy to be biased (even subconsciously) in making these
judgments.
To avoid such possible biases, it’s best to do this experiment “blind”.
In a blind
experiment, the scientist doesn’t know the identity of the sample that she/he is looking at.
One lab partner should pipet 10μL of a culture on to a microscope slide and cover with a
coverslip.
The second partner will view the cells under the microscope (I recommend 400x
magnification and phase-contrast illumination) without knowing which sample they’re looking at. For
each culture, view at least 100 cells and determine if each is a ‘normal’ cell or a shmoo. If needed,
refer to the drawings in your notebook from Week 2 for a reminder of what a ‘normal’ yeast cell looks
like. Calculate the percentage of cells in each of the four cultures that are shmooing. Draw a few
representative shmooing cells and non-shmooing cells in your notebook.
Once you have gathered all of your data record it on the clipboard at the front of the lab. Be careful
that you are recording the right thing with the right units: Abs420 units per milligram of total protein per
minute.
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After Lab:
• The class data will be collected and posted to the class website. Print the table and tape it into
your notebook.
• For each pair of cultures (with and without α factor) calculate how much the α factor increased
the β-galactosidase activity.
For example, did the hormone increase the amount of enzyme
1.8-fold, 3-fold or 10-fold?
• For each protein examined, classify it as either 1) needed to activate the pathway, 2) needed to
deactivate the pathway, 3) irrelevant to the pathway, or 4) it’s role is unclear. Briefly explain
why you made your decisions. Be sure to rely on both types of data (shmooing and FUS1
activation).
• Obviously, the pathway starts with α factor.
But its possible that the pathway divides
somewhere within the cell to lead to the different results of shmoo formation and FUS1
activation. Do any of the mutants separate the shmooing output from the FUS1 output? How
do you know?
• Can this sort of data be used to determine the order of proteins in a signal transduction pathway?
If so, what’s the order? If not, what else do we need to do to determine the order?
• Your notebook is due at the beginning of the final exam, 1:00 p.m. Monday, March 11th. No
late notebooks will be accepted.
Acknowledgement:
Thanks to Barbara Hoopes (Colgate University) for building pBH315. Experiments using this plasmid
to study signal transduction in the undergraduate laboratory were originally published at
http://www.zoo.utoronto.ca/able/volumes/vol-19/5-hoopes.pdf
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