Development of a Fluorescence-Based Approach to Visualize Subunit Stoichiometry and Protein-Protein Interactions at the Single-Molecule Level

Biological systems contain complex networks of protein interactions, making it difficult to discern the role of specific proteins using commonly employed macromolecular experimental techniques. The aim of my project was to minimize these complications by developing protocols to investigate the oligomeric state of proteins and visualize protein-protein interactions in vivo at the single-molecule level, thereby providing a clearer understanding of the structure and interaction of these molecules. I first set out to study TaALMT, a membrane transport protein shown to be involved in mediating abiotic stress responses in plant roots. To determine its stoichiometry, i.e. the interaction among TaALMT1 subunits, cRNA of TaALMT tagged to Green Fluorescent Protein (GFP) was microinjected into Xenopus laevis oocytes, and the heterologously expressed fusion-protein was imaged using Total Internal Reflection Fluorescence (TIRF) microscopy. The inherent property of GFP to photobleach after extended exposure to its excitation wavelength, in conjunction with the resolution of TIRF microscopy, allowed me to visualize discrete decreases in fluorescent intensity as each molecule of GFP lost its ability to emit light. The number of photobleaching steps is directly correlated to the number of individual interacting GFP-tagged protein subunits. Analysis of these experimental results revealed that in vivo TaALMT predominantly exists in the dimeric state. In addition, I implemented the same approach to determine protein-protein interactions using a two-color imaging strategy. To achieve this, I designed and constructed vector backbones to express CIPK2::mScarlet in X. oocytes. In macromolecular studies, this kinase was previously shown to interact with the plant membrane transporter AtMATE1. By simultaneously expressing AtMATE::GFP and CIPK2::mScarlet in X. oocytes, I was able to validate their protein-protein interaction at the single-molecule level using TIRF microscopy. In the future, this novel methodology that I have developed can be used into study the oligomeric state and protein-protein interactions of new proteins-of-interest, providing key insight into their fundamental interactions in vivo at the single-molecule level.

My Experience

The Plant Genome Research Program helped me grow as a rising professional in the scientific community and furthered my understanding of work in a research setting. As a member of the Piñeros lab, I was able to develop a broad scientific skill set while creating tools to study proteins at the single-molecule level. Namely, I gained experience with gene cloning, nucleic acid extraction and isolation, protein expression in Xenopus laevis oocytes, TIRF microscopy, and data analysis. While using these techniques, I also learned how to effectively solve research-based problems and apply critical thinking to refine experimental methodologies. Furthermore, the valuable guidance I received from my mentor not only helped me become a better scientist but also a better collaborator, communicator, and engaged member of the scientific community. His advice, kindness, and expertise made me more confident in my ability to work in a research environment and reaffirmed my pursuit of a scientific career. This research project, which was primarily conducted to benefit scientists, also made me aware of the challenges and importance of conveying its results to the public. Now, having completed the science communication training in this program, I feel better prepared to participate in meaningful scientific discussions with a broader audience. Going forward, I want to incorporate these new skills and principles into my work as I continue pursuing a career in research.