Objectives ✔ Apply proper technique in using, handling, and storing a microscope

Objectives
✔ Apply proper technique in using, handling, and storing a microscope
✔ Be able to prepare different specimens
✔ Know signal transduction pathway and predict
General Instructions
1. These experiments must be done in pairs.
2. Lab will be completed only when instructor signs it.
3. To get an instructor’s signature you need to present all slides, drawings and to answer all required questions.
Signal transduction: Saccharomyces cerevisiae
One pathway that has been extensively studied as a model for understanding signal transduction in general occurs in baker’s or brewer’s yeast, Saccharomyces cerevisiae. This single-celled organism can exist in two different haploid “cell types” called a and α cells. In the absence of the other cell type, each type will divide mitotically, proceeding through one full cell cycle in about an hour. Such vegetatively dividing cells are easily distinguished microscopically. Actively dividing cells appear as large spheres with smaller or equal-sized spheres budding from their periphery. If the two cell types, a and α cells, are in close proximity, they can mate by fusing to form a diploid cell. Mating between two cells requires the following things:
1. arrest of the cell cycle in the G1 phase before DNA is synthesized.
2. changing cell shape (called “shmooing”) to bring the cells closer together.
3. increasing transcription and translation of genes involved in cell fusion.
How does an a cell know there is an α cell in the vicinity with which it can mate? The α cells release a 13 amino acid peptide “factor” 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 all three of the effects above. At the same time a cells also produce α factor which binds to an a factor receptor on α cells.
Binding of α factor to the α-factor receptor on the surface of a cells activates a G-protein complex that in turn activates a kinase complex. The activated kinase complex turns on a protein kinase enzyme that then phosphorylates two proteins, the cell arrest factor and the transcription factor. In response to these two phosphorylated proteins, cells arrest at the G1 phase of the cell cycle and transcription of genes involved in mating is initiated (Figure 1).
Fig1. Representation of the signal transduction pathway of mating in a type cells of yeast (Saccharomyces cerevisiae). α factor is a 13 amino acid peptide released by the opposite mating type cell, α cells. Binding of α factor to the α factor receptor on the surface of a cells activates a G-protein complex which in turn activates a kinase complex. The activated kinase complex turns on a protein kinase enzyme which then phosphorylates two proteins, the cell arrest factor and the transcription factor. In response to these two phosphorylated proteins, cells arrest at the G1 phase of the cell cycle and transcription of genes involved in mating is initiated.
Cell cycle in yeast
The cell cycle is a combination of the stages through which a cell passes from one cell division to the next. The cycle is divided into the M phase (which includes mitosis and cytokinesis), and interphase. Interphase is subdivided into three stages: a growth period (G1) prior to, and in preparation for, DNA replication , a synthesis stage (S) when DNA is replicated and a second growth stage (G2) that precedes cell division.
S. cerevisiae is a “budding yeast” that divides by growing a bud on the cell (Figure 2). These cells can be easily seen through a microscope.
Fig. 2. Budding in the cell cycle of yeast (Saccharomyces cerevisiae). Budding begins in the S phase of the cell cycle and is completed after mitosis with the separation of the mother and daughter cells.
When cells are in G1, they do not contain a bud. When they enter S phase, they begin budding. As they progress through the cell cycle, the bud grows until, at the end of the G2, the cell looks like a “dumbbell”. During the M phase the bud separates from the cell to give two new cells. The percentage of unbudded cells is a measure of the number of cells not progressing through the cell cycle, but arrested in G1. Cell morphology can tell you something else. It can tell you if cells are responding to the complementary mating factor (Figure 3). Shmooing is a sign that cells are receiving the signal and that they are increasing transcription of mating specific genes.
Fig. 3. Photograph of budding (a) and “shmooing” yeast cells. The cell on the left is budding and the one on the right is “shmooing”. Budding occurs in the haploid cells of yeast and is the nonsexual method of reproduction. “Shmooing” is one of the responses to the signal transduction pathway initiated in a haploid yeast cell by a mating factor from the opposite mating type. It allows the cells to come together and fuse to form a diploid
This exercise will examine the effect of α factor on a cells.
PROCEDURE
Materials:
1. Microscope
2. Slides
3. Cover slips
4. Markers
5. Yeast strains:
WT- wild type (stimulated with α-factor and unstimulated)
far1- mutation in the arrest factor (stimulated α-factor and
unstimulated)
ste2 – mutation in the receptor (stimulated α-factor and unstimulated)
Experimental design:
Each pair should prepare slides for each strain (stimulated and unstimulated).
1. Label a slide (for example: W/stimulated)
2. Prepare a pipet to transfer 20uL
3. Shake a tube with yeast culture
4. Take 20uL of yeast culture and add to the labeled slide
5. Put cover slip on the top (see appendix 2 for instructions)
6. Observe the specimen under the microscope (see appendix 1 for instructions)
7. Record the number of budded cells and “shmooing” cells in Table 1
8. Draw and label your observation
9. Repeat this procedure for each of six yeast cultures (3 cultures per person)
10. Make a conclusion about how α factor influences the presence of unbudded cells