You are interested in the mobility of two different unknown integral plasma membrane proteins—protein X and protein Y—in a cell. In one cell, you fluorescently label all the X proteins

Question

In another cell, you label all the Y proteins. When you perform FRAP analysis on both cells, you find that most of the protein X fluorescence has recovered within 3 minutes, but recovery of the protein Y fluorescence is significantly slower. Even after 10 minutes, only 50% of the protein Y fluorescence has recovered. Describe the FRAP experimental protocol, explain what the results tell you about mobility of the X and Y proteins, and propose a possible explanation for why the mobilities of the two proteins differ.
What will be an ideal response?

Answer 1

Answer: For Fluorescence Recovery After Photobleaching (FRAP), the fluorescence of proteins in a spot on the membrane is bleached with a laser beam and recovery of fluorescence in that spot is measured over time. Recovery occurs when the proteins with photobleached labels diffuse out of the spot and proteins with unbleached labels diffuse in. Rapid recovery of protein X fluorescence indicates that protein X readily diffuses in the plasma membrane with little to no constraint. The slower and incomplete recovery of protein Y fluorescence indicates protein Y diffusion is constrained. Protein Y diffusion could be constrained, because the protein is associated associated with other membrane constituents in large complexes or lipid rafts, or it could be associated with extracellular or intracellular peripheral structures such as the extracellular matrix or the cytoskeleton.