INTRODUCTION: By using synthetic biological materials to assemble structures, process information, and harness energy, it is becoming possible to engineer new structures, such as artificial organelles. Towards this goal, our recent reports focus on protein polymers that reversibly phase separate into microdomains within live cells. Depending on their peptide sequence, they stimulate sorting or assembly of co-expressed proteins. We also developed microdomains that specifically intervene in clathrin-mediated endocytosis. METHOD: Elastin-like polypeptides (ELPs) are protein polymers, of the sequence (VPGXG)l that self-associate above a transition temperature, which depends on X and l. Utilizing ELP phase separation, we hypothesized that receptor tyrosine kinases can be reversibly clustered, thus modulating the activity of these important trans membrane proteins. In response to ligand, epidermal growth factor receptors (EGFR) auto-phosphorylate, internalize, and signal for cell survival. Due to its importance in cancer proliferation and the availability of clinically relevant kinase inhibitors, human EGFR was used to test this hypothesis. EGFR morphology was assessed as a function of temperature using both live and fixed cell imaging assayed by indirect immunofluorescence and fluorescent protein tags respectively. RESULT: A small library of EGFR ELP fusions was characterized, many of which display temperature-dependent changes in morphology. Most notably, phase separation was associated with the clustering of EGFR fusions into microdomains that rapidly internalized within the cell. These microdomains can be visualized during live cell imaging using a co-transfected green fluorescent protein fused to a related ELP. Most importantly, these internalized microdomains showed evidence of phosphorylation. CONCLUSION: Via genetic engineering, it is possible to express ELP fusions with trans membrane proteins that gain the ability to phase separate as a function of temperature. With additional study, this technology may become a tool for the controlled manipulation of biological signaling pathways. These tools may enable the dissection of cellular signaling pathways, the high throughput selection of small molecule inhibitors of desirable specificity, and the engineering of synthetic biological components for cellular therapies. ACKNOWLEDGEMENT: The National Institute of Health R21EB012281 to J.A.M., and P30 CA014089 to the Norris Comprehensive Cancer Center.

Artificial organelles: Cellular expression and assembly of polypeptide microdomains

249th National Meeting (2015)

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