Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Virginia A
Amanda Clark - Auburn University
Co-Author(s): Abby Beatty & Tonia Schwartz, Auburn University, Auburn, AL
Insulin-like growth factors (IGFs) are key hormone peptides regulating the Insulin and Insulin-like signaling (IIS) pathway, a pathway required for growth, metabolism, and reproduction. IGFs and other key proteins in the IIS pathway are highly conserved across vertebrate lineages including reptiles, but there are still gaps in our knowledge about the function of the IIS pathway and its members in reptiles [1,2]. Previous work has shown that although IGF2 is highly conserved across reptiles, IGF1 has experienced diversifying selection across the reptile clade. This is in contrast to mammals where IGF1 is under purifying selection . Substantial amino acid diversity in IGF1 between green anole (Anolis carolinensis) and brown anole (A. sagrei) lizards is concentrated in a domain associated with IGF1 Receptor binding affinity [2,3]. In mammalian in vitro cell cultures, IGFs are known to be fibrogenic/mitogenic and involved in cellular proliferation [4,5]. Cell culture provides a model for studying physiological and biochemical function and preliminary insight on cellular and possibly organismal response to drugs, growth factors, and/or stressors, but optimization of this method and associated culture assays in non-model organisms is not well documented [6,7]. We describe the establishment of three fibroblast lines from A. sagrei (brown anole lizard) tail tips and their use in characterizing the function of reptilian IIS pathway with species-specific recombinant IGFs expressed in and purified from E. coli. To optimize and assess prolific response to IGF treatments, cells were seeded and synchronized before serial time- and dose- dependent exposure to recombinant brown anole IGF1 and IGF2 treatments. Cellular proliferation in response to IGF dose and exposure time was assessed via growth curve analysis and BrdU assay. We then test the bioinformatic prediction that the amino acid sequence variation in IGF1 between green and brown anoles has a functional effect on cell proliferation via binding to the IGF1 receptor through the application of brown or green anole IGF1 or IGF2 to culture wells. Cellular proliferation in response to the hormones was directly assessed via growth curve analysis and indirectly via cell metabolism assays, after time- and dose-optimized exposure to peptide treatments. Expression of transforming growth factor-?1 (TGF-?1), cytokine expressed in response to IGF stimulation in human dermal fibroblasts, was quantified via qPCR . Results to be discussed include insights and challenges of primary culture with non-model ectothermic organisms, functional verification and use of lab purified recombinant proteins from non-model organisms, and cellular response to IIS activation by IGF proteins in the context of known genetic sequence variation and conservation patterns.
Funder Acknowledgement(s): Funding provided by Auburn University. A. Clark is also supported by the National Science Foundation Graduation Research Fellowship Program.
Faculty Advisor: Tonia S. Schwartz, email@example.com
Role: A. Clark contributed the experimental design, cell cultures, data analysis, and writing. A. Beatty contributed IGFs T. Schwartz contributed to the experimental design, statistical analysis, and coordinated writing.