Author ORCID Identifier
Date of Award
Dr. Susan Bane
Science and Mathematics
Organic compounds—Synthesis; Cancer cells; Pancreas; Tubulins
Microtubules and the desire to visualize them has been of utmost importance in understanding the behavior of the cytoskeleton. Even further, scientists are using this knowledge to elucidate cancerous activities as they occur within cells. In order to acquire this knowledge, the following methods have been established: the expression of tubulin after it has been fused with a fluorescent protein, the insertion of tubulin that has been exogenously labeled, and the use of fluorescent-probe conjugated microtubule ligands, such as paclitaxel. These methods, however, tend to distort the protein’s native function, thereby preventing an accurate analysis. This concern drives the need for an innovative approach that does not involve bulky attachments or genetic manipulation. One proposed approach is through the exploitation of specific, innate cellular processes, such as the post-translational modification responsible for the tyrosination of α-tubulin. By labeling α-tubulin with a tyrosine derivative capable of reacting with a fluorescent probe, the cellular activities pertaining to cancer can effectively be monitored.
In order to test this approach, 3-formyltyrosine (3FY) was synthesized and ligated in vitro to the α-tubulin subunit of microtubules in prostate cancer cells (PC3s) via the post-translational tyrosination facilitated by tubulin tyrosine ligase (TTL). A functionalized fluorophore, 3-trifluoromethyl-7-hydrazinyl coumarin (TFCH), was chosen as a means to optimize this technique which had previously been done with coumarin hydrazine. Coumarin hydrazine, although effective in labeling, mostly absorbs in the UV range and photo-decomposes easily. TFCH proves a better fluorophore for this process since it both absorbs at 405 nm and exhibits greater photostability.
Although there was no definitive conclusion regarding TFCH’s effectiveness in 3 microtubule visualization, the conditions and experimental procedure were further elucidated in such a way that should hopefully eliminate possible pitfalls in the future. Thus, this study serves as a foundation for the following studies to explore TFCH, as well as other fluorophores, both in vitro and in living cells.
Bonacasa, Angelina, "The synthesis of 3-formyltyrosine for the purpose of in virto fluorescent visualization of microtubules" (2022). Undergraduate Honors Theses. 18.