Question

• Your advisor is explaining his latest results in your weekly lab meeting. By fusing his protein of interest to GFP, he has shown that it is located entirely in the nucleus. But he wonders if it is a true nuclear protein or a shuttling protein that just spends most of its time in the nucleus. He is unsure how to resolve this issue. Having just read an article about how a similar problem was answered, you suggest that he make a heterocaryon by fusing cells that are expressing his tagged protein with an excess of cells that are not expressing it. You tell him that in the presence of a protein synthesis inhibitor to block new synthesis of the tagged protein he can resolve the issue by examining fused cells with two nuclei. He gives you a puzzled look and asks how does that help. You tell him what he has so often told you: “Think about it.

  • How would examining the two nuclei in a heterocaryon answer the question? What results would you expect if the protein were a true nuclear protein? What would you expect if it were a shuttling protein? B.

  • Why did you suggest that a protein synthesis inhibitor would be needed in this experiment? 3.

Answer 1

A heterokaryon is a cell which has two or more nuclei of different origin in a common cytoplasm. For example, heterokaryon can be formed by fusing mouse and human cells.

Non-shuttling proteins may accumulate in nucleus due to nuclear import pathway which is unidirectional. These are true nuclear proteins. Other proteins can shuttle between nucleus and cytoplasm as they can be targeted simultaneously by nuclear import and export pathways.

Heterokaryon assays can differentiate between nuclear proteins that are shuttling or non-shuttling. If the protein is a shuttling protein and is present only in donor cells, it will appear in the receptor cell. However, if it is true nuclear protein, then it cannot be exported out of the donor nucleus. Hence, it will never be seen in the receptor nucleus.

In the experiment, the GFP tagged protein was present in the donor cells, while the receptor cells lacked this protein. The receptor cells are grown in presence of leptomycin. Leptomycin B is an inhibitor of nuclear export. It binds to CRM1/ exportin 1. The donor and receptor nuclei are differentiated on basis of shape or expression of different fluorescence tags other than GFP.

The heterokaryon would therefore have two nuclei, the donor nucleus and the receptor nucleus. Assessment of expression of protein can be performed by fluorescence/confocal microscopy. If the protein is true nuclear protein, GFP expression will be seen only in donor nucleus. If the protein is shuttling protein, then its expression will be seen in both donor and receptor nucleus. As the receptor nucleus is in incapable of nuclear export, the protein cannot come out of the receptor nucleus.

Protein synthesis/ translation inhibitors need to be added, in order to ensure that no new protein synthesis can occur of this protein in the cytoplasm. This ensures that the protein that shuttles to receptor nucleus is from the donor cell nucleus.