Advancing Single Molecule Methods to Understand Translation Regulation in Live Cells

Cells use one third of their energy to produce proteins. Thus, cells require to precisely control protein production. Current knowledge of protein production regulation is based on mixing the contents of millions of cells to measure the average protein production rates. However, we now know that individual cells can behave differently than population averages. This research program will develop and apply new methods that allow measuring protein production in individual cells to determine when and where the regulatory events happen. These methods are based on measuring the speed of individual ribosomes (the protein production machines of the cell) in live cells. Evaluating thousands of ribosomes allows discerning when and how protein production, as well as other key functions of ribosomes such as UV or high temperature responses. More broadly, the research team will introduce high school students to the use of microscopy to study cellular and molecular biology processes. It will also develop courses to provide the basic skills needed for imaging of live cells, and how to use these skills to further our understanding of the fundamental principles of cellular and molecular biology. Live-cell biosensors have revolutionized our understanding of signaling and cell cycle regulation by enabling biochemical characterization of individual cells as they respond to environmental perturbation. However, many important biochemical parameters remain inaccessible through live single-cell approaches. Single-molecule tracking (SMT) approaches have allowed researchers in the chromatin regulation field to quantify the amount of DNA-bound vs. free proteins in live single cells. This is because proteins diffuse slowly when they are bound to DNA. The PI’s team has recently discovered that many other proteins, including translation factors and signaling proteins, show qualitatively distinct diffusion properties depending on the size and properties of the complex they are bound to. Thus, measuring the diffusion properties of thousands of single-molecule trajectories in live cells allows to qualitatively increase the number of biochemical parameters that can be measured at the single cell level. The specific objectives are: 1) Dissect global translation dynamics in live single cells, 2) Dissect global translation dynamics during UV radiation-induced ribotoxic stress response (RSR), and 3) Implement an educational plan to (a) expose high school students to imaging based cellular and molecular biology ,and (b) develop a graduate course elective to provide the basic skills and knowledge required for live cell imaging based molecular and cellular biology. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.