Question

In a separate line of experiments, you are studying a protein that you suspect shuttles between the nucleus and the cytoplasm. However, when you localize a GFP-tagged fusion protein, it appears to be only in the nucleus. To test whether it shuttles, you set up a heterokaryon assay. You take a cell expressing the GFP fusion protein and partially fuse that cell with another that does not express the GFP protein. The result is a heterokaryon that has a single cytoplasm but two separate nuclei. You treat the heterokaryon with the translation inhibitor cycloheximide for several hours and then examine the localization of the GFP protein. What do you predict to see if the GFP protein shuttles between the nucleus and the cytoplasm? Briefly explain your answer.

Answer 1

Heterokaryons are cells that have to or more nuclei obtained from different origins. When cell expressing GFP tagged protein (donor cell) is fused with a cell that does not express GFP tagged protein (recipient cell), a heterokaryon cell is obtained that has two nuclei (donor and recipient nuclei) sharing a common cytoplasm. If there is nucleus to cytoplasmic shuttling, then GFP protein should exit the GFP nuclei, get exported to cytoplasm and will then be imported into the nucleus that lacked GFP protein expression (recipient nucleus). Thus, now the recipient nucleus will now start expressing the GFP protein. If the GFP protein does not exhibit nucleus-cytoplasm shuttling, then it will not be expressed in recipient nucleus over time. Protein shuttling from nucleus to cytoplasm can occur either by just diffusion through nuclear pores or via a receptor mediated pathway. The GFP protein is only expressed in nucleus indicating a receptor mediated pathways. If protein diffuses through nuclear pores, it would also be seen in cytoplasm, which is not the case. If it is shuttling between the nucleus and cytoplasm, it should have nuclear import and export signals.

Cycloheximide is a drug that inhibits elongation step of protein translation. Thus, it will inhibit new protein synthesis in the heterokaryon cells. This will allow only old preexisting protein (before Cycloheximide treatment) to be visualized in the heterokaryon cell. As this is a shuttling protein, the preexisting GFP protein will shuttle from the nucleus to the cytoplasm and from cytoplasm to the recipient nucleus. If there is high rate of shuttling, then both donor and recipient nucleus will show expression of the GFP protein. Non shuttling proteins will not leave the donor nucleus and hence, would only be expressed by the donor nucleus. Cycloheximide treatment is performed to prevent new protein expression in cytoplasm of the GFP protein.