Probelm Set 2 Spring 2024

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Feb 20, 2024

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Problem Set 2 Principles of Cell Biology Spring 2024 1. Roger Tsien won the Nobel Prize for discovering and developing the GFP technology which has contributed yet more “tools” that we can add to our cell and molecular biology “tool box” to address contemporary research challenges in cell and molecular biology. a. Why is “reporter molecule” an apt description for the GFP family and what is the difference between a constitutive GFP reporter and a regulated GFP reporter? A reporter molecule is a good description since GFP is used to track genes in cells. A regulated GFP reporter can turn on and off while a constitutive GFP reporter is always turned on and labels cells permanently b. How was the constitutive reporter important in the two “humanized kidney” reports of 2017 and 2023 discussed in class? GFP was used to track the progress of humanized pig stem cells growing, which successfully created a human kidney. The constitutive reporter tracked the humanized genes, and the scientists were able to see that the stem cells remained in the pig and didn’t move around the organism. They achieved their sole purpose, the growth of a human kidney inside another animal. 2. a. What was the rationale for both of these experiments? These experiments were used to see if there’s a possible solution to the organ shortage. If the kidney can be successfully transplanted into a human and work efficiently, then kidneys and other organs can be grown in rapid succession. Less people would die while waiting on the UNOS transplant list. b. A 1/31/24 report on CNN details a “humanized pig heart” that was transplanted into a recipient who was ineligible for a heart transplant because of peripheral vascular disease. (https://www.cnn.com/2024/01/30/health/pig-organ-transplant- xenotransplantation/index.html ) How does “compassionate use” apply to this transplant? Compassionate use applies to this transplant since it was the patient’s last hope. Since they were unable to be on the transplant list for a human heart, their only choice was to go home to a sure death or to try a humanized pig heart, which could possibly work. Because the patient didn’t have much hope, compassionate use was allowed. 3. We have emphasized in this section of the course that most techniques and laboratory devices don’t arise de novo (anew) but rather evolve from pre-existing technologies. For instance, the first plate reading spectrofluorometer was developed by RVB while a consultant for Millipore Corporation given the need to quantify cells stained with vital fluorescence markers so that an average emission intensity of all the cells present in a sample could be expressed quantitatively rather than limited to the only analysis method available prior to that time which was to simply qualitatively visualize the labeled single cells one by one using fluorescence microscopy. FRET is yet another example of this evolutionary process where one technology gives rise to another.

a. What is the rationale of FRET evolving from the GFP family? FRET, Forester Resonance Energy Transfer, uses GFP to see what happens. Specifically FRET is used to see when two molecules are getting close together and GFP helps us to determine this. b. You are doing a FRET experiment and find that the two proteins that are labeled with GFP and YFP don’t exhibit FRET in conditions where they should exhibit FRET fluorescence due to their 1 to 10 nm proximity. Why is this most probably the case? Maybe the fluorescence proteins aren’t turned on or phosphorylated? c. What is the difference between FRAP and FRET? FRAP relies on photobleaching to achieve its goal in finding the membrane fluidity of cells. The photobleaching leaves a dark spot which is easy to monitior. FRET, on the other hand, doesn’t use photobleaching. It solely relies on the fluorescence proteins. d. Challenge : We mentioned “biosensor” in class and detailed in one case how a select biosensor, calmodulin, can measure a change in intracellular calcium by fluorescing. How does a calmodulin biosensor work and how would this be useful if you were interested in analyzing the synchronized beating of a living human cardiac organoid in culture? A biosensor is used to see the cell physiology and when calcium binds to proteins. It uses GFP to highlight the binding sight/ protein. This is useful for analyzing a cardiac organoid since it can monitor whether the amount of calcium binding to the proteins is sufficient or comparable to a regular human heart. 4. Below are some experimental scenarios that were discussed last week during lecture and in your text. Based on the description identify the technique used in each case. a. You are using an indirect mAb technique with two different primary and secondary antibodies with the latter labeled with 5 nm and 20 nm gold particles respectively to identify two separate proteins in tissue sections. Double label ultrastructural immunocytochemistry Ultrastructural Immunocytochemistry b. You intend to view the internal topology or structure of cell membranes relying on freezing the cells and ultimately viewing a carbon stabilized platinum replica in a microscope. Cryosections/ Freeze fracture c. You are transfecting a culture of CHO (Chinese Ovary Cells) with a GFP labeled gene of interest using an approach that encapsulates the latter for delivery to the cells. FRET liposomes

d. You are working in a laboratory that does Somatic Cell Nuclear Transfer (SCNT) and need to inject a somatic nucleus into an enucleated (nucleus removed) egg cell. PALM? Single cell intracellular injection with a micropipette e. You are working in a blood lab and are checking patients for their number of T cells using a mAb that binds specifically to T cells and not other blood cell types. This system can also separate these T cells from other blood cells for subsequent molecular analysis. ELISA? FACS f. You are doing an experiment with two fluorochromes using a microscope that can achieve much better practical limit of resolution compared to either a point- scanning or spinning disk confocal microscope. TEM Super resolution microscopy g. You are doing an experiment whereby you need to purify a cell fraction that contains the outer cell membrane (smooth microsomes) and separate it from rough microsomes which are resealed membranes originating from the rough endoplasmic reticulum. Detergent mixed micelle Density gradient centrification Rate somal centrification h. You are separating groups of proteins using a column that contains positively charged beads which ensures that the proteins being separated retain their native activity once eluted. This column separates these proteins based on the differences in overall negative charge of the proteins. Selective surfaces Ion exchange chromatography i. You want to identify a specific mRNA of interest in neurons using a fluoresceinated probe that is complimentary to this mRNA of interest to see if it preferentially localizes in the neurons’ dendrites. FISH j. You are working in an oncology lab examining human liver tissue sections and find a cell of unknown type which you decide to remove from the section with the help of a laser and analyze at a later time using single cell analysis techniques. Laser capture microdissection microscopy

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k. You fix some tissue with glutaraldehyde and osmium tetroxide and subsequently infiltrate the sample with epoxy plastic for subsequent sectioning and staining with lead and uranium. TEM, plastic thin sectioning technique l. You are attempting to isolate and purify an unknown “orphan” receptor to “Ligand X” the latter of which you have purified and now covalently bound to non-charged beads that are packed in a column chromatography column. Ion exchange chromatography Affinity chromatography

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