Examination Candidate Number: Desk Number: UNIVERSITY OF YORK BSc Stage 2 Degree Examinations 2017-18 Department: BIOLOGY Title of Exam: Cell Biology Time allowed: 1 hour and 30 minutes Total marks available for this paper: 80 This paper has two parts: Section A: Short answer questions (40 marks) Answer all questions in the spaces provided on the examination paper Section B: Problem questions (40 marks) Answer all questions in the spaces provided on the examination paper Instructions: The marks available for each question are indicated on the paper A calculator will be provided For marker use only: 1 2 3 4 5 6 7 8 9 10 11 Total as % DO NOT WRITE ON THIS BOOKLET BEFORE THE EXAM BEGINS DO NOT TURN OVER THIS PAGE UNTIL INSTRUCTED TO DO SO BY AN INVIGILATOR page 1 of 13
SECTION A: Short answer questions Answer all questions in the spaces provided Mark total for this section: 40 1. a) Compare and contrast paracellular and transcellular transport. (2 marks) b) Give an example of where in the body each is used. (2 marks) c) Discuss how key features of tight junctions regulate paracellular and transcellular transport across epithelial tissue. (4 marks) page 2 of 13
2. The figure below represents a flow cytometry analysis of a mixed population of cells: a) Annotate the figure to indicate which phases of the cell cycle are represented by the labels A, B and C. (3 Marks) b) If the population of cells above were normal human cells, what ploidy would the cells in phase A and phase C be? (2 Marks) c) The cells above are a mixed population, but it is often useful to synchronise a population of cells so that they are all in the same phase of the cell cycle. Name two ways of achieving natural synchrony of the cell cycle in cultured mammalian cells. (2 Marks) page 3 of 13
3. a) What would the effects of N-ethylmaleimide be on membrane traffic? (1 mark) b) Give an example of a protein tethering complex. (1 mark) c) If a synaptic vesicle membrane contains Qa, Qb and R SNAREs, what SNARE classification type would be required for a functional SNARE complex and in what membrane would it have to be for neurotransmitter release? (2 marks) 4. a) Which amino acid side chain typically provides a ubiquitin acceptor site within a polypeptide? (1 mark) b) If a mutation in a cell caused the rate of deubiquitination to be higher than ubiquitination, what would be the consequences on: i) mitochondria (2 marks) ii) Epidermal growth factor receptor (2 marks) page 4 of 13
5. Outline the molecular mechanisms involved in Fas-receptor mediated destruction of influenza-infected cells. (5 marks) 6. Draw and label the basic structure of a type I collagen molecule. (3 marks) page 5 of 13
7. An experimental protocol is being designed to differentiate induced pluripotent stem cells (ipscs) into corneal tissue to help treat impaired eyesight. a) Provide one advantage and one disadvantage of using ipscs compared to adult limbal stem cells. (2 marks) b) Part of the protocol involves growing the ipscs on different extracellular matrix (ECM) substrates, namely type IV collagen and heparan sulfate proteoglycans. Give reasons why these particular ECM substrates were chosen. (3 marks) c) How would you determine whether the ipscs had differentiated into corneal cells in vitro? (3 marks) page 6 of 13
SECTION B: Problem questions Answer all questions in the spaces provided Mark total for this section: 40 8. You have developed an assay to follow formation of actin filaments from purified actin monomers. Your assay starts with purified actin monomers labeled with a fluorescent probe. Upon polymerization the fluorescence of the probe increases, allowing polymerization to be measured. Data from a typical time course is shown and plotted on the left hand graph. The right hand graph shows the equilibrium distribution of actin in free subunits (monomers) and in filaments, as a function of actin concentration. a) While optimizing conditions for the assay you establish a requirement for a physiological buffer (ph7.4) and the addition of another molecule in order for the actin monomers to form filaments. What is that molecule and why is it required here? (2 marks) b) Speculate as to why the intensity of fluorescence at time zero is not zero. (1 mark) page 7 of 13
c) What stages of actin filament assembly do the three phases of the polymerization curve on the left hand graph reflect? (3 marks) d) Estimate the critical concentration of actin polymerization for this experiment. (1 mark) e) How would the curve on the left change if you doubled the initial concentration of actin monomers used in the assay? Would the concentration of free (monomeric) actin at equilibrium be higher or lower than in the original experiment? (4 marks) f) At 1.4 mg/ml pure tubulin, microtubules formed from 13 protofilaments grow at a rate of 2 μm/min. At this growth rate how many αβ-tubulin dimers (8 nm in length) are added to the ends of a microtubule each second? (4 marks) page 8 of 13
9. A patient arrives in the clinic and examination of the patient s blood identifies high levels of the lysosomal hydrolase cathepsin-b. a) Suggest two possible explanations for this observation. (2 marks) b) The cathepsin-b protein from the patient and a normal individual were subjected to SDS-PAGE and transferred to two membranes. One membrane was western blotted for cathepsin-b (image on the left) and the other membrane was incubated with a mannose-binding lectin and the lectin visualised (image on the right). Explain the observations and how this causes elevated cathepsin-b in the patient s blood. (4 marks) c) When cells from the patient were treated with rapamycin, a drug which can induce autophagy, no effects were seen on the availability of nutrients. Given also the data above, suggest what might be wrong with the patient. (3 marks) page 9 of 13
10. You incubate adipose cells with insulin, which promotes exocytosis of the glucose transporter GLUT4. The extracellular concentration of glucose, before insulin addition, is 10 mm. The cells after insulin addition, remove 50% of the extracellular glucose per minute. a) Plot a graph of extracellular glucose concentration vs time for the 5 minutes after the addition of insulin. (4 marks) page 10 of 13
The binding of insulin to its receptor causes the receptor to be endocytosed and degraded by the lysosome. To increase insulin sensitivity and subsequently glucose uptake, cells were treated with various compounds with the aim to inhibit insulin-receptor endocytosis. Glucose uptake was measured as above. The following table shows the drugs used and the rate of glucose uptake. Compound t ½ glucose uptake (min) Dynasore (inhibits dynamin) 0.5 Nocodazole 1 Vanadate (phosphatase inhibitor) 2 SP600125 1 Pitstop-1 (clathrin inhibitor) 1 b) If you assume that the glucose uptake is wholly dependent upon the levels of the active cell-surface insulin-receptor what can you conclude about how the insulin-receptor is endocytosed? (5 marks) page 11 of 13
11. In an experiment, you continuously label all proteins in a batch of sea urchin eggs with a radioactive label and take samples every 10 minutes after fertilisation. You then run these samples on a gel using electrophoresis. You see a band ( BAND X ) that increases in intensity up to the 40 minute time point, but then rapidly and significantly reduces in intensity (indicated by the square brackets and label). The result is in the figure below: a) Give two possible reasons why BAND X drops in intensity after 40 minutes (when the sea urchin eggs enter M phase). (2 Marks) b) What is the most likely name of the protein represented by BAND X? (1 Mark) page 12 of 13
You repeat the experiment, but this time you label the cells every ten minutes. You take samples and process them as before. You see a band ( BAND Y ) which represents the same protein as BAND X. However, this time the band is present at the same intensity at all time points. The result is in the figure below: c) What is the name of this labelling technique? (1 Mark) d) Explain why the different labelling techniques produce different results. (2 Marks) e) Given the results of the second experiment, which of the reasons given in (a) for the drop in intensity in the first experiment is correct? (1 Mark) page 13 of 13