assignment
Questions Page | 2 Instruction: You are provided with 4 scientific papers and questions associated with specific figures from each paper. Please read the whole of the paper carefully. Answer the questions specifically but use your understanding of the whole paper to inform your answers. Paper 1: Phosphorylation triggers presynaptic phase separation of Liprin-α3 to control active zone structure. (20 Marks total) An excellent example of cross talk and integration of cell signalling is at the synapse. Here calcium and cAMP signalling regulate a cascade of kinases and phosphatases which in turn control neurotransmitter release. A presynaptic protein complex consisting of proteins like liprin, Munc13-1, RIM and bassoon interact with each other and interact with key effectors of synaptic transmission such as the synaptic vesicles and calcium channels. How proteins within the complex interact largely unknown and is an exciting field of research. This paper, Emperador-Melero et al 2021 presents evidence that phosphorylation of liprin-α3 by protein kinase C (PKC) regulates liprin-liprin interactions which then drive organisation of the presynaptic complex and regulate neurotransmitter release. Question 1 (5 Marks) From the data in these two panels what can you conclude about the action of PKC on liprin-α3 distribution? (summarise in 5 dots points, 5 marks) Taken from Figure 1 Page | 3 Question 2 (2 Marks) This panel shows super resolution (STED) images at a synapse to provide evidence that PKC phosphorylation affects synapse structure. Answer the following: Question 2a Why do the authors include immunostaining for Bassoon? (1 mark) Questions 2b How can you tell, from these images, whether liprin-α3 phosphorylation drives these changes? (1 mark) Taken from Figure 3 Page | 4 Question 3 (3 Marks) The authors produce a double knock out (KO) mouse that removes liprin-α2 and liprin-α3. Figure 5 summarises the functional effect of this on the synapse. The recordings shown are the post-synaptic current responses. Think of these are “reporters” for the presynaptic function – if this is compromised the (post synaptic) currents will be smaller. This panel shows post-synaptic currents in the knock-out and in “rescue” experiments where liprin-α3 has been re-expressed. Answer the following: Question 3a What is the intention of using the mutant liprin-α3 where serine 760 has been replaced with glycine? (1 mark) Question 3b What do the post-synaptic current traces in panel t tell us? (1 mark) Question 3c What do the immunostaining data in panel v and w tell us? (1 mark) Taken from Figure 5 Page | 5 Question 4 (5 Marks) Figure 6 defines the functional effects of liprin-α3 on the spontaneous miniature excitatory postsynaptic currents (mEPSC). Each downward deflection in panel a is an mEPSC and after treatment with PMA the frequency of the mEPSCs increases. Summarise the data shown in these panels (a, b, c) and the conclusions that can be drawn. (5 marks) Taken from Figure 6 Page | 6 Question 5 (5 Marks) Here, using immunostaining and super resolution microscopy the authors show the effects of PKC on the structure of the presynaptic complex. In this panel they are looking specifically at Mun13-1. Answer the following: Question 5a In the knock out animals (KO) does anything happen? If so, what? (2 mark) Question 5b Can you conclude from this data that liprin-α3 phosphorylation is important? If so, what are the evidence? (3 marks) Taken from Figure 7 Page | 7 Paper 2: Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport. (20 Marks total) Several structures of prokaryotic homologues of the glutamate transporter family (SLC1A) have been solved and reveal details about the conformational changes required for transport and the transport mechanism of this family. But detailed understanding of the human transporters requires structures and functional analysis. This paper describes three cryo-EM structures of the human glutamate transporter EAAT3 that were identified from one sample of protein and reveals details about substrate and coupled ion binding and confirms that the human glutamate transporters use the same transport mechanism as the prokaryotic homologue, GltPh. Question 1 (7 Marks) Supplementary Figure 1 D (Figure S1D) demonstrates that aspartate-activated currents are only observed in the presence of Na+, agreeing with previous literature that EAAT3 (like all neurotransmitter transporters) relies on the Na+ gradient across the cell membrane to drive transport of substrate against a concentration gradient. 1. What other ions is transport via EAAT3 coupled to? (2 marks) 2. What is the name of this type of transport process that is coupled to the co-transport of ions? (1 mark) 3. Name the transport protein that is responsible for maintaining the Na+ gradient across the membrane (1 mark) and how explain how it achieves this? (3 marks) Question 2 (10 Marks) Page | 8 Figure 1D reveals two of the structures that were resolved of EAAT3 in this study. 1. Briefly describe these two conformational states (2 marks) and explain the type of transport mechanism used by EAAT3 (and all glutamate transporters) (4 marks)? 2. Name and briefly describe one other mechanism that membrane transporters use to move molecules across the membrane (3 marks). Give an example of a transporter that uses this mechanism (1 mark). Page | 9 Question 3 (3 Marks) In this paper, the authors claim that “inward-facing hEAAT3g has a remarkably low substrate affinity, at least under our imaging conditions”. What evidence do they present that supports this claim (2 marks) and how is this different to what we know about the prokaryotic homologue, GltPh (1 mark). Page | 10 Paper 3: Membrane cholesterol dependence of cannabinoid modulation of glycine receptor. (20 Marks total) Cannabinoids exert therapeutic effects by modulating the activity of a variety of membrane proteins. One of these targets is the glycine receptor (a member of the pentameric ligand-gated ion channel family of ion channels). Cholesterol is a major component of all membranes and has been shown to directly, and indirectly, regulate the activity of many membrane proteins. This paper uses a combination of biochemical methods, electrophysiological methods and molecular dynamics simulations to demonstrate that membrane cholesterol plays an important role in the mechanism of cannabinoid modulation of glycine receptors. Question 1 (10 Marks) 1. Describe the evidence that glycine receptors associate with cholesterol-rich domains of the cell membrane (Figure 1)? (2 marks) 2. Describe the experiment that shows that cholesterol is required for THC potentiation. As part of you answer, identify the negative control used in the experiment. (Figure 1) (3 marks) 3. Based on your knowledge of the Glycine Receptor (from the lecture notes), why do you think that α1β GlyRs and neuronal GlyRs do not show as much potentiation as α1 GlyRs and α3 GlyRs? (Figure 1). What can you say about the likely composition of GlyRs found in spinal cord neurones. What would expect to see for α2 GlyRs. (5 marks) Page | 11 Question 2 (2 Marks) 1. What was the effects of cholesterol depletion on activation of α1 GlyRs expressed in HEK293T cells? (Figure 2) (2 marks) Page | 12 Question 3 (8 Marks) 1. Cholesterol depletion has similar effects on THC, anandamide and cannabidiol enhancement of GlyR function, but this is not observed for propofol, isoflurane or ethanol. Suggest reasons why propofol, ethanol and isoflurane are not affected by cholesterol (Figure 4). (2 marks) 2. The effects of cholesterol on ligand binding to GlyR was simulated over 50 ns using two different membranes: 1. Pure POPC and 2. POPC+cholesterol in a ratio of 5/1 (Methods section). Suggest two ways that you could improve these simulation conditions to provide a more realistic prediction of GlyR function in a neuronal membrane? (6 marks) Page | 13 Paper 4: G protein-coupled receptor (GPR)40-dependent potentiation of insulin secretion in mouse islets is mediated by protein kinase D1. (20 Marks total) It is well established that acutely free fatty acids potentiate glucose induced insulin secretion from beta-cells and this effect is mediated by activation of the free fatty acid receptor GPR40 on the surface of the beta-cells. when beta-cells are acutely exposed to free fatty acids the fatty acid receptor, GPR40. In this paper the authors have shown that this effect of GPR40 on insulin secretion is mediated by protein kinase D1. Question 1 (4 Marks) 1. Based on the data presented in Figure 1 what is the evidence that oleate potentiation of glucose induced insulin secretion is GPR40 dependent? (2 marks) 2. What is the reason the authors have concluded that oleate potentiate only the second phase of insulin secretion and not the first phase? (2 marks) Page | 14 Question 2 (7 Marks) 1. Describe the key changes you see between WT and KO islets. (5 marks) 2. What was the reason for including Latrunculin treatment in these experiments? (2 marks) Page | 15 Question 3 (6 Marks) 1. Since exogenous DAG could potentiate glucose stimulated insulin secretion both in the WT and GPR40 KO islet, what does this result say on whether DAG acts upstream or downstream of GPR40? (2 marks) 2. Explain how the Figure 4g is instrumental in helping the authors draw the conclusion that the effect of oleate phosphorylation of PKD-1 is GPR40 dependent. (4 marks) Page | 16 Question 4 (3 Marks) 1. Describe the key findings from this figure and describe the importance of Latrunculin treatment. (3 marks) Paper 1-4/.DS_Store __MACOSX/Paper 1-4/._.DS_Store Paper 1-4/Paper 1.pdf 1 1 2 Phosphorylation triggers presynaptic phase separation of Liprin-α3 to control active 3 zone structure 4 5 6 7 8 9 Javier Emperador-Melero1, Man Yan Wong1, Shan Shan H. Wang1, Giovanni de Nola1, Tom 10 Kirchhausen2, and Pascal S. Kaeser1,# 11 12 13 14 1. Department of Neurobiology, Harvard Medical School, Boston, MA 02115 15 2. Departments of Cell Biology and Pediatrics, Harvard Medical School and Program in Cellular 16 and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115 17 18 19 20 #correspondence and lead contact:
[email protected] 21 22 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It