Are Both Fus3 Kinase and Formin Required for Directional Cell Growth During Mati

Question

Are Both Fus3 Kinase and Formin Required for Directional Cell Growth During Mating in Yeast? When a yeast cell binds mating factor molecules from a cell of the opposite mating type, a signaling pathway causes it to grow a projection toward the potential mate. The cell with the projection is called a “shmoo” (because it resembles a 1950s cartoon character by that name). Researchers sought to deter- mine how mating factor signaling leads to growth of this cell projection on one side of the cell—in other words, to asymmetric cell growth. Pre- vious work had shown that activation of Fus3, one of the kinases in the signaling cascade, caused it to move to the membrane near where the mating factor bound its receptor. The researchers’ first experiment iden- tified one of the phosphorylation targets of Fus3 kinase as formin, a pro- tein directing microfilament construction. Based on this information, the researchers developed the working model shown here for the signaling pathway that leads to the formation of shmoo projections in yeast cells.

How the Experiment Was Done To determine if Fus3 and formin were required for shmoo formation, the researchers generated two mutant yeast strains: one that lacked the gene for making Fus3 kinase (a strain called Fus3) and one that lacked the gene for making formin (formin). To observe the effects of these mutations on schmoo forma- tion after cells’ exposure to mating factor, the symmetry of growth was investigated. First, the existing cell walls of each strain were stained with a green fluorescent dye. These green-stained cells were then exposed

to mating factor and stained with a red fluorescent dye that labels only new cell wall growth. Growth of the cell on all sides (symmetric growth) is indicated by a uniform yellow color, resulting from merged green and red stains. (This occurs normally in wild-type cells that have not been ex- posed to mating factor, which are not shown.)

Data from the Experiment The micrographs above, right, were taken of wild-type, Fus3, and formin cells after they were stained green, exposed to mating factor, and then stained red. The wild-type cells ex- pressed both Fus3 and formin.

Interpret the Data

1. A model helps scientists form a testable hypothesis. The diagram shows the working model of shmoo formation developed by the re- searchers. (a) What hypothesis from the model was being tested with the Fus3 strain? (b) With the formin strain? (c) What is the purpose of including wild-type yeast cells in the experiment?

When designing an experiment, scientists make predictions about what results will occur if their hypothesis is correct. (a) If the hypoth- esis about the role of Fus3 kinase activity in shmoo production is correct, what result should be observed in the Fus3 strain? If it is incorrect, what result is expected? (b) If the hypothesis about the role of formin in shmoo production is correct, what result should be ob- served in the formin strain? If it is incorrect, what result is expected?

3. For each micrograph, describe the shape of the cells and the pattern of cell wall staining. Explain the significance of your observations. Which strain(s) of yeast cells formed shmoos?

4. (a) Do the data support the hypothesis about the role of Fus3 kinase in shmoo production? (b) Do the data support the hypothesis about the role of formin in shmoo production? (c) Do the data support the working model (the working hypothesis) in the diagram?

5. Fus3 kinase and formin proteins are generally distributed evenly throughout a yeast cell. Based on the model in the diagram, explain why the projection would emerge on the same side of the cell that bound the mating factor.

6. What do you predict would happen if the yeast had a mutation that prevented the G protein from binding GTP?

SCIENTIFIC SKILLS EXERCISE using Experiments to Test a Model 1 Mating Mating Shmoo projection factor G protein-coupled acto forming activates Formin recepto Fus3 GTP subunit DP Phosphoryl- 2 G protein binds GTP becomes activated. ation Formin Formin cascade O Fus3 phos- Microfilament Fus3 Fus3 activating it. 6 Formin initiates growth of Phosphorylation cascade microfilaments that form activates Fus3, which moves the shmoo projections. to plasma membrane Are Both Fus3 Kinase and Formin Required for Directional Cel Growth During Mating in Yeast? When a yeast cell binds mating factor molecules from a cell of the opposite mating type a signaling pathway causes it to grow a projection toward the potential mate. The cell with the projection is called a "shmoo" (because it resembles a 950s cartoon character by that name). Researchers sought to deter- mine how mating factor signaling leads to growth of this cell projection on one side of the n other words, to asymmetric cell growth. Pre- vious work had shown that activation of Fus3, one of the kinases in the Wild type Aformin AFus3 signaling cascade, caused it to move to the membrane ne where the mating factor bound its receptor. The researchers' first experiment iden tified one of the phosphorylation targets of Fus3 kinas as formin, a pro- 2. When designing an experiment, scientists make predictions about tein directing microfilament construction. Based on this information, the what results will occur if the hypothesis is correct. (a) If the hypoth- researchers developed the working model shown here for the signaling esis about the role of Fus3 kinase activity in shmoo production is pathway that leads to the formation of shmoo projections in yeast cells. correct, what result should be observed in the AFus3 strain? If it is ncorrect, what result is expected? (b) If the hypothesis about the role How the Experiment Was Done To determine if Fus3 and formin of formin in shmoo production is correct, what result should be ob- were required for shmoo formation, the researchers generated two served in the Aformin strain? If it is incorrect, what result is mutant yeast strains: one that lacked the gene for making Fus3 kinase 3. For each micrograph, describe the shape of the cells and the pattern a strain called AFus3) and one that lacked the gene for making formin of cell wall staining. Explain the significance of your observations. (Aformin). To observe the effects of these mutations on schmoo forma Which strain (s) of yeast cells formed shmoos? ion after cells' exposure to mating factor, the symmetry of growth was nvestigated. First, the existing cell walls of each strain were stained with 4. (a) Do the data support the hypothesis about the role of Fus3 kinase a green fluorescent dye. These green-stained cells were then exposed shmoo production? (b) Do the data support the hypothesis a o mating factor and stained with a red fluorescent dye that labels only the role of formin in shmoo production? (e) Do the new cell wall growth. Growth of the cell on all sides (symmetric growth working model (the working hypothesis) in the diagram? is indicated by a uniform yellow color, resulting from merged green and 5. Fus3 kinase and formin proteins are generally distributed evenly ed stains. (This occurs normally in wild-type cells that have not been ex- throughout a yeast cell. Based on the model in the diagram, explain posed to mating factor, which are not shown.) why the projection would emerge on the same side of the cell that bound the mating factor Data from the Experiment The micrographs above, right, were taken 6. What do you predict would happen if the yeast had a mutation that of wild-type, AFus3, and Aformin cells after they were stained green, prevented the G protein from binding GTP? exposed to mating factor, and then stained red. The wild-type cells ex pressed both Fus3 and formin A version of this Scientific Skills Exercise can be assigned in Interpret the Data MasteringBiology. 1. A model helps scientists form a testable hypothesis. The diagram Data from D. Matheos et al Pheromone-induced polarization is dependent on the shows the working model of shmoo formation developed by the re- Fus3p MAPK acting through the formin p. Journal of CellBiology 165:99-1 searchers. (a) What hypothesis from the model was being tested with (2004 of including wild-type yeast cells in the experiment?

Explanation / Answer

1) a) The following hypothesis were tested from the model. The signal transduction pathway for the normal Shmoo formation requires Fus3.

b) The signal transduction pathway for the normal Shmoo formation requires formin.

c) The purpose of including wild type yeast cells in the experiment is to show the normal Shmoo formation under experimental conditions.

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