Question 1. (____/ 7 marks)

Chloroplasts, mitochondria, and nuclei are three intracellular organelles that are surrounded by two biological membranes. Although they possess some structural similarities, they have many              differences in function and biogenesis. For statements 1 to 7 in the table below, indicate whether it applies to:

A. Chloroplasts only

B. Mitochondria only

C. Nuclei only

D. Both chloroplasts and mitochondria, but not nuclei

E. All three organelles

Question 2.  (____/ 12 marks)

For each of the observations below, write a statement that could explain these results, based on what the data shows and on your knowledge of cellular structure/ function (3 marks each)

A. If taxol is added to cells at metaphase, the chromosomes do not move towards the spindle poles in anaphase.

B. Electron micrographs show that mitochondria in heart muscle have a much higher density of cristae than mitochondria in skin cells.

C. Microtubules formed in vitro from tubulin that is bound to a non-hydrolyzable form of GTP were found to be exceptionally stable.

D. Some membrane proteins can be readily extracted with 1M NaCl, whereas others require the use of a detergent.

Question 3. (____/15 marks)

On the domain maps provided, draw and label all targeting sequences that you predict would be   found in the proteins, A - E. If you predict no targeting sequence is required, clearly indicate this in your answer – a blank will NOT be considered for marks. (2 marks each):

A. Soluble ER resident

-

B. Rubisco Small Subunit 2B – a Chloroplast stromal

-

C. Transcription factor

-

D. α-tubulin

-

E. Glycophorin A (plasma membrane protein), 1 transmembrane domain, N-terminus outside the cell.

N-                                                                                                           -C

Match each of the proteins mentioned above with its cellular location, as identified in each micrograph (arrow) below. (1 mark each):

Question 4 (____/9 marks)

The epidermal growth factor receptor (EGFR) is a transmembrane protein localized to the plasma   membrane in skin epithelial cells. When EGFR is activated by binding to the extracellular peptide    EGF, it is internalized via vesicles. Ultimately, this internalization will lead to changes in cell growth and division. Researchers studied the relationship between EGFR internalization and cell growth by investigating wild type cells compared to cells with a non-functional mutant form of clathrin.

A. The first experiment examined the amount of EGFR found in early endosomes in the wild type and mutant cells, in the absence (-EGF) or presence (+EGF) of the extracellular peptide. Describe the results shown, and explain what you can conclude based on these data. (3 marks)

B. In the next experiment, they examined the effect of EGFR internalization on cell cycle proteins, to see whether there were any changes. The SDS-PAGE data below shows the results for Mitotic      Cyclin (M-Cyclin), a protein known to promote entry into mitosis, in the absence or presence of      EGF. Describe the results, and explain what you can conclude based on the gel shown. (2 marks).

C. The researchers discovered that after the wild-type cells are exposed to EGF, a transcription factor called ERK1/2 moves into the nucleus. On the other hand, ERK1/2 remained localized in the               cytoplasm when the clathrin mutants were exposed to EGF. Considering this and the rest of the data  in this question, propose a model describing how EGF-binding to EGFR might affect the cell cycle      progression. In your explanation, include the role of EGF and its receptor, clathrin, the transcription    factor ERK1/2 and Mitotic Cyclin. (4 marks)

Question 5 (____/ 10 marks)

Actin Capping Protein (ACP) binds to the plus end of actin filaments, preventing the actin filaments from gaining or losing monomers. Its activity is blocked by regulatory proteins such as V- 1. In this experiment, Takeda et al. (2010) examine the role of V- 1 and ACP in the regulation of actin polymerization.  In Panel A, the ratio of F-actin (actin filaments) to G-actin (actin monomers) were measured for WT (normal) and V1 (over- expresses V- 1) cells. In Panel B, actin filaments and nuclei were stained with fluorescent dyes and cells were examined through fluorescence and light microscopy. Assume that both cell lines express the same amount of ACP.

A. Compare the bars in the chart in Panel A. What do they tell you about the amount of actin in each cell type? How might the change in expression levels of V1 be affecting ACP’s ability to function?     (3 marks).

B. Panel B shows paired brightfield and fluorescence microscopy images of each of the cell types   measured in Panel A. Compare the shape of the WT cells to the V1 cells, and explain how the actin shown in the fluorescence images is contributing to that shape. (4 marks)

C. Based on the data shown in Panels A and B, how might V- 1 be influencing cell shape in the overexpressing cells? (3 marks)

Question 6 (____/ 8 Marks)

You generated a yeast strain with a temperature-sensitive mutated form of M-cyclin that is unable to fold stably at a non-permissive temperature (37°C). To assess its role in the cell cycle, you grow       these mutant cells in a test tube at the permissive temperature (25°C) then shift them to 37°C.

A. Explain what happens to M-cyclin concentration as the cell progresses through interphase at the permissive temperature. How does this compare at the non-permissive temperature? (2 marks)

B. What effect would this mutation have on the regulation of cell cycle at the non-permissive temperature (2 marks)?

C. You fluorescently labelled the DNA of your mutant yeast cells and used fluorescence activated cell sorting (FACS) to analyze the effect of this mutation on the cell cycle. Note that these cells complete  a cell cycle in 90 minutes where interphase lasts for approximately 70 minutes. In your experiment,    you have 2 tubes (labeled Tube 1 and Tube 2). In Tube 1, you incubate the yeast cells at the non-     permissive temperature, 37°C, for 2 hours. In Tube 2, you incubate the cells for 2 hours at non-          permissive temperature, followed by 30 minutes at the permissive temperature. In the appropriate      spaces below, draw the expected FACS readout from each Tube and label the graphs with the           relevant phase of cell cycle expected.

Question 7 (____/ 9 Marks)

Nocodazole is used as an anti-cancer drug that binds to a/b tubulin subunits inside cells.  Shown below are fluorescence images of cells in the presence or absence of nocodazole, by fluorescence-tagged immunolabeling for a-tubulin.

A. Describe the fluorescence pattern seen in Panels A and B. What does this tell us about the effect of nocodozole on microtubules? (3 marks)

B.  Based on your knowledge of microtubule polymerization at the plus end, propose a model for how Nocodazole might be leading to the results shown. (2 mark)

C. Predict what would happen to the cell in Panel B if the nocodazole was washed away and the cell was allowed to recover. (1 mark)

D. Predict the impact of nocodazole treatment on constitutive secretion in these cells? (1 mark) Explain why. (1 mark)

E. Propose a role for nocodazole in the M-phase which makes it useful as an anti-cancer drug. (1 mark)