8. You perform the following microscopy experiments to measure the signaling out
ID: 266714 • Letter: 8
Question
8. You perform the following microscopy experiments to measure the signaling output of two pathways and observe the following results. Experiment 1 - You genetically tag an M-cyclin with GFP. You add an upstream signal to a cell culture to activate the M-cyclin Result 1 - GFP fluorescence intensity increases. Experiment 2- You genetically tag adenylyl cyclase with RFP. You add an upstream signal to a cell culture to activate adenylyl cyclase. Result 2-There is no change in the RFP fluorescence intensity A. (3pts) Why would adding the respective upstream signals lead to changes in M-cyclin-GFP intensity but not adenylyl cyclase-RFP intensity? B. (3pts) For experiment 1, predict the timescale over which the GFP intensity increases.Explanation / Answer
Cyclins are cell cycle regulatory proteins and there are different cyclins where each one is activated in different phases of cell cycle. M-cyclin is activated during mitosis and its level start increasing when the cell enters the mitotic stage. When GFP (Green Flourescent Protein) is tagged with M-cyclin gene, the intensity of GFP increases when the M-cyclins level increase.
Adenylyl cyclase is an integral membrane protein present in the plasma membrane of the cell. It catalyzes the formation of cAMP (second messenger) from ATP, when it is activated by G-alpha subunit of G protein coupled receptor (GPCR). When the RFP (Red Flourescent Protein) is tagged to adenylyl cyclase gene, the intensity of RFP does not increase as it is an integral membrane protein which once synthesized and part of plasma membrane, its level remains the same.
Therefore, M-cyclin is secreted during mitosis phase and then degraded at the end of mitosis while adenylyl cyclase once synthesized remains in the plasma membrane.
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