Frost et al (2009) Propagation of Tau Misfolding from the Outside to the Inside
ID: 149707 • Letter: F
Question
Frost et al (2009) Propagation of Tau Misfolding from the Outside to the Inside of a Cell. J. Biol. Chem. 284, 12845-12852.
After read above, please answer 1 to 10 or more. This is for discussion, so I would like to get accurate answer for those.
Read the methods section and the figure legends carefully. If you simply quote an answer from the paper, beware! I will probe to make sure you understand what you are parroting!! Also, you may have to go to cited references to find out details about
Note: When I say “describe Figure X” it is important to state the question being addressed, how it was addressed experimentally (what did they do to address the question), what controls were used, what the authors concluded, and whether or not you agree with those conclusions (and why).
I urge you to read the Introduction first, then go to the Results section and as you read, go back to the Experimental Procedures to understand HOW they did the experiments described in Results. This is far easier than reading the Experimental Procedures in their entirety before reading the Results!!!!!
diagrams that support your verbal answer are very helpful!” are particularly suited for diagrams/pictures.
1. How were cell samples prepared for SDS-PAGE and Western blot analysis? What is the purpose of using trypsin? What does “triton-soluble” and “triton-insoluble” mean? What is a “whole cell lysate”?
2. Briefly explain SDS-PAGE and Western blotting. What are they used for and what are the underlying principles? PICTURES/DIAGRAMS SPEAK A THOUSAND WORDS….. diagrams that support your verbal answer are very helpful!
3. Briefly explain the immunofluorescence approach used in this study. What is confocal microscopy? What is a Z stack? PICTURES/DIAGRAMS SPEAK A THOUSAND WORDS….. diagrams that support your verbal answer are very helpful!
4. Still thinking about immunofluorescence. What did the authors have to do before they could visualize Tau-YFP or MTBR-YFP in cells? What was the purpose of fixation with methanol or paraformaldehyde? What was the purpose of tubulin staining? Why were MTBR monomers and aggregates labeled with AF488? What is AF488? What is “co-labeling” and why do it? What is the fundamental difference between using HA and YFP as fluorescent tags?
5. Explain the principle of flow cytometry. Briefly state how flow cytometry was used in this study. PICTURES/DIAGRAMS SPEAK A THOUSAND WORDS….. diagrams that support your verbal answer are very helpful!
Results:
6. Describe the constructs used to characterize the activities of Tau (Fig 1A). Please go through Figure 1A and explain it. What do the numbers, boxes and colors represent? How might a vector designed to express an exogenous protein in mammalian cells differ from one designed to express the same protein in bacterial cells?
7. Explain Figure 1B-F. What did the authors do? What did the authors conclude? Do you agree? Why or why not? What do the authors mean by “inclusions”?
8. What overall question were the authors addressing in the experiment shown in Figure 2. Explain Figure 2A and B. How does arachidonic acid induce fibrillation of MTBR-Tau? Why do the authors care if the recombinant MTBR-HA is sensitive to digestion by trypsin?
9. What question were the authors addressing in the experiment shown in Figure 2C-D? What did the authors do? What did the authors conclude? Do you agree? Why or why not?
10. What question were the authors addressing in the experiment shown in Figure 2E? What did the authors do? What did the authors conclude? Do you agree? Why or why not?
11. What overall question were the authors addressing in the experiment shown in Figure 3? What did the authors do? What did the authors conclude? Do you agree? Why or why not?
12. What overall question were the authors addressing in the experiment shown in Figure 4AC? What did the authors do? What is the significance of using the Tau5 antibody? What is the significance of “detergent insoluble” vs “detergent soluble” Tau-YFP? Why are these experiments important? What did the authors conclude? Do you agree? Why or why not?
13. What question were the authors addressing in the experiment shown in Figure 4D-E? What did the authors do? What did the authors conclude? Do you agree? Why or why not?
14. What overall questions were the authors addressing in the experiment shown in Figure 5AB? What did the authors do? What did the authors conclude? Do you agree? Why or why not? What questions were the authors addressing in the experiment shown in Figure 5C-D? What did the authors do? What did the authors conclude? Do you agree? Why or why not?
15. What overall questions were the authors addressing in the experiment shown in Figure 6? What did the authors do? Why did the authors transfect one population of cells with just mCherry? What did the authors conclude? Do you agree? Why or why not? When is a difference between treatments “significant”?
16. What overall questions were the authors addressing in the experiment shown in Figure 7? What did the authors do? What did the authors conclude? Do you agree? Why or why not?
17. How did the authors know that the “transfer” of Tau-YFP did not occur by cell fusion? Explain.
Discussion:
18. Propose a mechanism by which a Tau aggregate taken up by a cell via endocytosis could gain access to the cytoplasm of that cell. Why is this even a question? Think hard about your Intro and Cell Bio courses before you answer this one! PICTURES/DIAGRAMS SPEAK A THOUSAND WORDS….. diagrams that support your verbal answer are very helpful!
19. Paraphrase, or even better, draw a model, depicting the mechanism by which Tau proteins propagate protein misfolding within the brain. You can base this on the authors model or, even better, on your own! PICTURES/DIAGRAMS SPEAK A THOUSAND WORDS….. diagrams that support your verbal answer are very helpful!
Explanation / Answer
Please find the answers below:
Answer 1:
Part 1: How were cell samples prepared for SDS-PAGE and Western blot analysis?
Answer : The cell samples for SDS PAGE and western blotting were prepared under aseptic conditions by triton in phosphate buffered saline (PBS) in presence of protease inhibitory cocktail. The aggregates of cells were suspended in this buffer and allowed to lyse to release cytosolic proteins. This resulted in generation of two types of cellular proteins, soluble in triton and insoluble in triton. Both of these fractions were later investigated for different experimental procedures.
Part 2: What is the purpose of using trypsin?
Answer :Trypsin is a proteolytic enzyme generally used to lyse hard muscular tissue extracellular matrix such as skeletal muscles and myocardium. In cellular culture, trypsin is utilized to de-adhere the cells from a fixed substratum such as a growth flask or plate to degrade the cellular adherence proteins so that the cells might detach from the growth plate. In the present study, 0.25% of trypsin was used for this purpose.
Part 3: What does “triton-soluble” and “triton-insoluble” mean?
Answer : Triton is a strong non-ionic detergent and surfactant used for solubilization of proteins. This agent is generally used for separation of ionic and non-ionic proteins on the basis of their solubility. The triton-soluble proteins are non-ionin proteins and insouble proteins are ionic proteins. Thus, by differential centrifugation of total cellular proteins in triton, a fraction of proteins can be obtained.
Part 4: What is a “whole cell lysate”?
Answer : Whole cell lysate is a cumulative term used to demonstrate obtaining a total fraction of all the cellular proteins from all the possible organelles such as cytosol, nucleus, lysosomes, mitochondria, endoplasmic reticulum etc. This term is used when the investigation about protein expression is independent of their origin and the gross effect of a regulatory molecule is being investigated on the whole cell. Generally, harsh cellular lysis methods are used to lyse all the membranes inside the cell to release all the proteins.
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