PhD Defense: Yue Guo
Visualizing dynamics of the central dogma in living single cells
Imaging the central dogma at single molecule resolution in single living cells reveals the spatial and temporal dynamics and ubiquitous cell-to-cell variability of molecular events and provides quantitative insight into gene expression regulation. SunTag-based translation reporter enabled real time measurement of translation activity on individual polysomes. However, most approaches require cell fixation or have limited sensitivity for live-cell applications in imaging transcription. I develop SunRISER (SunTag-based Reporter for Imaging Signal Enriched mRNA), a computationally and experimentally optimized approach for unambiguous detection of single mRNA molecules in living cells. When viewed by epifluorescence microscopy, SunRISER-labeled mRNAs show strong signal-to-background and resistance to photobleaching that together enable long-term mRNA imaging studies. SunRISER variants using 8x and 10x stem-loop arrays, demonstrate effective mRNA detection while significantly reducing alterations to target mRNA sequences. I also characterize SunRISER to observe mRNA inheritance during mitosis and find that stresses enhance diversity among post-mitotic sister cells. Taken together, SunRISER enables a window into living cells to observe aspects of the central dogma in addition to roles of mRNAs in rare and dynamical trafficking events.
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