The End? Mutant Receptors and Oncogenesis

It’s hard to believe my summer of research has come to an end. In my last blog post two weeks ago, I had just observed my first set of TRa1 transfected HeLa cells under fluorescence microscopy. Since then, I have continued to work with my two TR mutants, hcc-TRa1 and tc-TRa1, performing replicate experiments and testing for statistically significant differences in localization patterns.

Back in my first post, I explained how each replicate experiment takes about a whole week to complete. The HeLa cells must be first trypsinized, incubated for a 24-hour period, transfected with a lipofection reagent, and then incubated for another 24 hours. Then, they are fixed with formaldehyde, mounted in a microscope slide and stained for fluorescence; after yet another 24-hour period, the cells are ready to be analyzed under fluorescence microscopy. As I explained in my last blog post, the Allison lab scores TR cellular localization by calculating the ratio of protein fluorescence in the nucleus to the cytoplasm (N/C Ratio). Following their protocols, I scored each of my TRa1 variants in my three replicate experiments and then performed an unpaired (two sample) t-test between the data sets for the wild type receptor and each of the mutant variants.

Results show that with a p-value of <0.05 for all data sets, I successfully rejected the null hypothesis. There IS a significant difference in the localization patterns of these TR variants. Mutant hcc-TRa1, tc-TRa1, and the co-transfected wild type TRa1 had a significantly lower N/C Ratio, indicating that the protein had a much more cytosolic distribution than normal. The amino acid substitutions in the NLS-2 of hcc-TRa1 and tc-TRa1 may be the principal cause of these irregular shuttling patterns, but it does not fully explain why the wild type TR also behaves differently when co-expressed alongside these mutants. Is as if some type of interaction is taking place between the receptors, leading the wild type to follow a similar intracellular localization pattern as its mutant cousin. This is particularly concerning when discussing TR’s role as a tumor suppressor; if the single presence of one of these mutations is enough to disrupt the behavior of the healthy allele, then there is an increased risk for oncogenesis and other pathologies. After seeing these results, I cannot help but wonder what are the molecular interactions taking place between wt-TRa1, hcc-TRa1 and tc-TRa1 leading to these abnormal observations?

 

What exactly is going down there?

Co-expression of GFP-tagged Wild Type TRa1 and mCherry-tagged tc-TRa1; we can see most of the protein surrounding the nuclei (blue) of the cells. Image taken in 7/21/17.

 

The summer may be over but my desire to understand these little proteins is not, which is why I am very excited to continue working with thyroid hormone receptor this coming fall in Dr. Allison’s lab. I hope to study further the interactions between wild type and mutant TR’s; moreover, I hope to understand how exactly hcc-TRa1 and tc-TRa1 are linked to oncogenesis. Is it only a matter of disrupting TR’s normal localization patterns? Or is some other mechanism responsible for leading the cells to these diseased states? I cannot wait to find out.

I also want to take this moment to thank the Charles Center for allowing me to develop my project this summer! It was an incredible opportunity to explore the field of molecular biology and I was very fortunate because I ended up loving the field. I also want to thank Dr. Lizabeth Allison and Vincent Roggero for all of their help and mentoring this past month; I cannot wait to continue working alongside you.

Thanks again to everyone for reading!

 

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