Cell Communication and Cell Division
Objective Understand the parts and effects of a signal transduction pathway and predict effects of disrupting parts of the pathway Part 1 Signal transduction affecting cell division in Saccharomyces cerevisiae Baker’s yeast (Saccharomyces cerevisiae). can exist in two different types called a and α cells. They are capable of reproducing in two ways. 1. Mitosis: In the absence of the other cell type, each type will divide mitotically (asexual), about once an hour. Mitotically dividing cells appear as large spheres with smaller or equal-sized spheres budding from their edge. Budding yeast 2. Mating- If the two cell types, a and α cells, are near each other, they can mate by fusing (sexual) to form a diploid cell. Mating between two cells requires the following which are caused by alpha factor signaling: 1. arrest of the cell cycle in the G1 phase –no mitosis (stop budding). 2. increasing transcription and translation of genes involved in cell fusion -includes schmooing to bring the cells closer together. Schmooing yeast
How does yeast reproduce?
How can you tell if yeast is reproducing in each of the two ways?
Part 2 Signaling responsible for yeast mating How does an a cell know there is an α cell near to it available for mating? The α cells release a 13 amino acid peptide called α factor. This peptide binds to a receptor, α factor receptor, on the cell surface of the a cells. Binding of the α factor to the receptor results in stopping mitosis (budding) and starting schmooing. factor signaling pathway 1. Binding of α factor to the α-factor receptor on a cells activates a G-protein complex 2. G protein complex activates a kinase complex. 3. Activated kinase complex phosphorylates two proteins: (a)cell arrest factor and (b)transcription factor. 5. (a)Phosphorylated cell arrest factor stops cell division (stops budding) (b)Phosphorylated transcription factor increases transcription (starts schmooing)
2 Show/describe what will happen to a type of yeast in the presence and absence of alpha-factor?
Part 3 Signaling pathways and the effects of disrupting the pathway. Below is an example signaling pathway and a demonstration of the effects of activating the signaling pathway. Mutations occuring in an enzyme in the signaling pathway can disrupt the effects of activating that pathway. What do you predict are the effects of the two mutations below on the activation of enzymes 4 5 and 6? Experiment: This “lab” will examine how disruption (mutation) of parts of a signaling pathway affect the results of activating that pathway. Specifically you will examine the effect of α factor on cell division in normal a cells (WTwild type) AND examine the effect of mutations that block the function of: 1. The α factor receptor (STE2 yeast) 2. The cell arrest factor (FAR1 yeast)
Predictions 1. Based on the signaling pathway described, what do you expect normal (wild type) yeast cells to do in the presence and absence of alpha-factor (bud/schmoo/both/neither)?
2. What do you expect type yeast cells that have no arrest factor (FAR2) to do in the presence and absence of alpha-factor (bud/schmoo/both/neither)? 3. What do you expect type yeast cells that have alpha-factor receptor (STE1) to do in the presence and absence of alpha-factor (bud/schmoo/both/neither)?
Sample: WT,WT stimulat ed , far1 , far1 stimulat ed , ste2, ste2 stimulat ed.
So what is
Cells preparing to divide mitotically(bu dding)
Cells preparing for sex reprod (shmooing)
Cells in G0/G1 (NOT shmooi ng or buddin g)
% of buddin g cells
% schmooi ng
For each one
This is a table for cell counting .
The lab report should have
Title Page: On an individual sheet of paper, include the number and title of the lab exercise, the name of the course, your name, and the date on which you submitted your report.
Abstract: A single paragraph summary of the entire report.
Must state the study’s purpose, a general approach, and the results/conclusion.
There should be no literature citations in an Abstract.
Limit Abstract to a maximum of 250 words.
This section should always be written last.
Introduction: 1-2 paragraphs introducing background information and the study’s goal.
Include background information needed for a reader to understand the study.
State the goal & hypothesis (in the form of a prediction statement) of your study.
Material and Methods: Concise description providing enough information to repeat experiments.
Must be written in paragraph form. DO NOT LIST YOUR MATERIALS.
Include times, volumes, etc; Omit what you used to measure with.
Incorrect example: “Solution A, Solution B, a micropipette, and tips were collected.
Using the micropipette, 500uL of Solution A was transferred into 500uL of Solution B. The
tip was then disposed of”.
Correct example: “500uL of Solution A was transferred into Solution B”.
Results: States and presents the data you collected. Include figure (s) and refer to the figures in the text.
Example 1: “as seen in Figure 1, the bacteria grew faster on glucose…”
Example 2: “…a significant increase was seen (Figure 1).”
Each figure must have a title, and appropriate labels and axis legends should be included,
so that each figure may stand alone.
Do not discuss what the data means.
Discussion: The discussion reiterates the hypothesis, the results, and what they mean.
Briefly repeats the data and discusses what they mean in context of the hypothesis.
States any mistakes/difficulties/future changes to the experimen
