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...

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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
Answered 5 days AfterJun 18, 2021

Answer To: Questions Page | 2 Instruction: You are provided with 4 scientific papers and questions associated...

Dr. Sulabh answered on Jun 19 2021
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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) The question is about the action of PKC on liprin distribution – you don’t seem to talk about that.
· The figure panel 1b. indicates the localisation of the liprin-(3 protein after the induction of the protein kinase C enzyme by the substrate named PMA (phorbol-12-myristate-13-acetate) and without the addition of the PMA substrate. The protein kinase C signaling pathway is activated after addition of PMA substrate. PMA is a strong activation of protein kinase C signaling pathway.
· The protein liprin-(3 formed fused protein aggregate after the addition of the PMA substrate which causes the activation of the protein kinase C enzyme.
· HEK293T cells were used for transfection with the control vector mVenus and with the other vector mVenus-Liprin-(3 vector.
· In the first panel of figure 1b there is a depiction of the expression levels of the mVenus-Liprin-(3 in the HEK293T cells along with a comparison of the levels of mVenus vector along after transfection in the HEK293T cell line without the addition of PMA substrate. 
· The levels of liprin-alpha3 proteins increase after the addition of PMA due to the addition of the protein kinase C enzyme thus causing the activation of the signaling cascade. The levels of lipirin-alpha3 protein are less without the addition of the substrate PMA in the HEK293T cell lines. The second panel of figure 1b indicates the expression levels of the liprin-alpha3 protein during the stage of washout. In diagram 1c of the given paper panel, there is an analysis of the percentage of transfection of mVenus-liprin-alpha3 vector with and without PMA addition and after a washout. In the mVenus-Liprin-alpha3 vector there is maximum efficiency of transfection after the addition of PMA and the efficiency of transfection is less if PMA is not added. The levels of transfection and expression of only the mVenus vector are less with and without the addition of PMA.
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)
The protein Bassoon is involved in the formation of the synapse in the neurons with the release of the neurotransmitter from the neuronal junctions. A protein complex is formed near the neurotransmitter site consisting of the different proteins like Bassoon, RIM, Munc 13, Liprin-(. Therefore, the protein Bassoon is included in immunostaining.
Questions 2b How can you tell, from these images, whether liprin-α3 phosphorylation drives these changes? (1 mark)
The experimental studies with the analysis of the synapsis formation with the generation of the active vesicles involve the contribution of Bassoon protein and Synaptophysin protein. The studies on the dead mutant of liprin-(3 protein revealed that phosphorylation is essential for the formation of the separation of the different vesicle layers or zones during the process of neurotransmitter release. The fluorescent images in the diagram reveal the formation of the different protein complexes with merging of liprin-(3 phosphorylated with Basoon, RIM, Munc13-1 and the synaptophysin proteins. The merging of the proteins is evident after the addition of PMA substrate.
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)
The replacement of Serine-360 in liprin-(3 protein with glycine leads to the generation of the dead mutant which confirmed the experimental result that the neruons synapse phase separation with the neurotransmitter secretion is due to the Serine-360 amino acid phosphorylation and protein kinase C activation mechanism of the liprin-(3 protein. The mutant liprin-(3 protein inhibits the process of neuronal phase separation and the activation of the enzyme protein kinase C is also inhibited.
Question 3b What do the post-synaptic current traces in panel t tell us? (1 mark)
The post synaptic current traces in the panel t indicate the distance of the separation of the protein Gephyrin with the other proteins like RIM, Munc13-1, RIM-BP-2 and the caveolin proteins in the presynaptic and the postsynaptic neurons. The current peak indicates the intensity of a protein and the separation of the current peak indicates the interaction and distance between the different proteins in the control and the knockout liprin-(2 and liprin-(3 protein neurons.
Question 3c What do the immunostaining data in panel v and w tell us? (1 mark)
The panel v in the diagram signifies the interaction of the Bassoon, RIM and the synaptophysin protein with merging in different samples of the knockout liprin protein and dead mutant liprin protein. In the knockout liprin and knockout liprin and the dead mutant liprin interaction with the other proteins, there is a phase separation of the different peaks as compared to the overlapping of the different phases in the liprin protein with the other proteins panel. The panel diagram w indicates the interaction of RIM protein with the other proteins during the formation of the synapse in the knockout liprin, knockout liprin+ liprin-(3 and in the knockout with the dead mutant liprin protein with statistical analysis.
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 a 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) I think u need say about the conclusion that can be drawn for each.
Conclusion:
· a. The conclusion for panel a in the figure 6 indicates the minimum excitatory postsynaptic current for the liprin-(3 protein in the knockout+ liprin-(3 neuron and in the knockout with the dead mutant of liprin-(3 protein. The addition of PMA increases the postsynaptic current peaks.
· b.The conclusion for panel b indicates the frequency of the generation of the current postsynaptic peaks in the knockout+ liprin-(3 neuron in the knockout with the dead mutant of liprin-(3 protein neuron before and after the addition of the PMA substrate.
· c.The conclusion for panel c indicates the percentage of the intensity of the postsynaptic peak after the addition of PMA in the knockout+ liprin-(3 neuron and in the knockout with the dead mutant of liprin-(3 protein neuron. The postsynaptic current peak intensity is more in the knockout liprin-(3 protein as compared to the liprin dead mutant neuron.
Taken from Figure 7
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)
In the knockout model as given in the microscopy image there is separation of the different current phases and there is hindrance in the formation of the synaptic vesicles and there is hindrance also in the release of the neurotransmitter from the neurons. In comparison the current peaks are resolved and there is the formation of the synapse in the knockout liprin and the liprin-(3 neurons. In the knockout and dead mutant liprin neuron also there is hindrance in the formation of the synapse, hindrance in the release of the neurotransmitter and separation of the current phases.
Question 5b Can...
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