May
31

Colocalization Experiment: GFP-vErbA and mCherry-rc6-TRα1

The mutant thyroid hormone receptor I have been studying, rc6, has a phenotype similar to that of a well characterized oncogenic protein: vErb-A. vErb-A is a highly mutated form of thyroid hormone receptor alpha that is linked to cancer formation. rc6 and vErb-A are both mutated forms of thyroid hormone receptor alpha. The mechanism by which vErb-A promotes cancer formation is well understood and could provide insight on the affects of rc6 in healthy cells. rc6 is not well characterized, as of now, but similarities between localization, dominant-negative activity, and aggregation suggest a similar mechanism of cellular disruption to vErb-A. rc6 is not proven to cause cancer but it is found in a substantial amount of renal clear cell carcinomas, a type of kidney cancer.

Characterizing rc6 may reveal any role that this mutant plays in renal clear cell carcinomas. Below are images from a recent pilot study looking at the colocalization between rc6 and vErb-A. Colocalization would indicate that both proteins either interact with each other or interact with the same cellular components. This data is subjective but promising. It appears to show the colocalization of rc6 and vErb-A in a HeLa (human) cell. Further trials and analysis will reveal if and to what degree these two proteins colocalize. If colocalization is occuring, further experiments will be necessary to determine the significance of colocalization. These experiments could include staining for aggresomes, comparing the rate of nuclear trafficking between the two mutants, or comparing the long term consequences for the cells when the mutants are expressed.

 

Comments

  1. cdong03 says:

    Hi,

    I’m glad that your preliminary study is going well! Since I’m not very familiar with your research I do have a few questions. Are both rc6 and vErba-A expressed in normal HeLa cells, or are they transfected in your experiment only? I also did some colocalization analysis myself a while back, though with embryos; do you have any data that indicates the degree of colocalization between the two proteins? Also, what is the magnification of your images? In addition, I’m wondering is there’s any rc6 knockdown/knockout studies done on renal clear cell carcinoma cell lines, or just overexpression in other cells?

    Good luck with your experiment!

    Chen

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