Real-time dynamics of gene expression at single molecule level
We have recently developed a new imaging technique called "SunTag" (Tanenbaum et al., 2014, Cell), which allows us link many GFPs to a protein molecule or organelle of interest (Fig. 1A). This GFP multimerization approach makes the fluorescence tags much brighter than was previously possible, and enables us to visualize complex biological processes with single molecule sensitivity in real-time in living cells. Using the SunTag technology, we can continuously monitor translation of single mRNA molecules in space and time (Yan et al., 2016, Cell).
We are employing SunTag technology to visualize gene expression control in living cells with incredible precision to uncover how regulatory mechanisms function at the molecular level, and how regulation of protein expression affects cell fate decisions. We are using a combination of quantitative single cell and single molecule fluorescence microscopy and computer simulations to look beyond cell population averages, and study how single cells tune gene expression over time.
Figure 1. Single molecule imaging of mRNA translation using the SunTag system. (A) Schematic of the SunTag fluorescence labeling strategy. (B) Imaging translation on single mRNA molecules in live cells.
Single-molecule analysis of viral infection, translation, replication, and virus-host interactions
RNA viruses are among the most prevalent pathogens and are a major burden on society. For example, coronaviruses can cause global pandemics, but even relatively mild viruses, such as common cold-causing rhinoviruses, have a big impact on our economy. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. The first hours are particularly relevant to study as the outcome of the infection may be determined at the early stages of an infection: how can the infected host cell detect an infection and how can the antiviral defense mechanisms of the host cell prevent spreading of an infection?
We have developed a single-molecule imaging assay based on our SunTag technology, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells (Tanenbaum et al, 2020, Cell). Using VIRIM we can detect the first moment of infection and follow viral replication in single cells. We are employing VIRIM to uncover the heterogeneity in infection, replication, and host cell responses between single cells . Based on a combination of single-molecule imaging of viral dynamics and host cell antiviral responses, we aim to identify the Achilles heels of various RNA viruses, which can serve as a starting point for the development of targeted therapies against virus infections.
Figure 2. Live-cell imaging of single viral RNA molecules. (A) Schematic of the translation and replication of individual RNA viruses in live cells. (B) Virus infection real-time imaging (VIRIM).
Manipulation of gene expression using synthetic transcription factors
Control of gene expression is critical for cell fate and homeostasis, and is often de-regulated in diseases like cancer. To study the function of gene expression regulation, it is critical to be able to perturb it. However, modulating the expression of endogenous genes has been very challenging. Recently, in collaboration with the lab of Jonathan Weissman at UCSF, we have developed a new system to modulate transcription rates using an artifical, precisely controlled transcription factor based the the CRISRP/Cas9 protein fused to transcription activation domains through the SunTag (Fig. 3) (Tanenbaum et al., 2014, Cell).
We are using this new technology to study how transcriptional regulation drives key cell cycle transitions, and how transcriptional control interplays with other gene expression regulatory mechanisms, like mRNA stability and translation.
Figure 3. Gene activation using synthetic transcription factors. (A) A nuclease-dead CRISPR/Cas9 protein is fused to an array of transcriptional activation domains through SunTag and targeted to an endogenous gene promoter to turn on transcription. (B) K562 cells in which the membrane receptor CXCR4 (red) is turned on in the bottom two cells using CRISPRa. (C) K562 cells in which CXCR4 gene activity is artifically modulated show increased cell migration.
Post-transcriptional regulation of the cell cycle in single cells
Hundreds of proteins show altered expression as cells progress through the cell cycle, but the mechanisms underlying these changes remain poorly understood. While a significant body of work has focused on regulation of protein degradation, very little is known about the control of protein synthesis, even though protein levels are equally dependent on protein degradation and synthesis. Protein synthesis rates can be regulated through many different regulatory mechanisms, including transcriptional and translational control, mRNA localization and mRNA stability. We have developed new techniques to visualize different steps in gene expression in single cells to understand how these different mechanisms that alter gene expression activity are controlled as cells progress through the cell cycle (Tanenbaum et al., 2015, eLife). Using these techniques, we aim to understand how protein levels are modulated over time and how control of gene expression ensures reliable cell cycle decisions in single cells.
Figure 4. Single molecule FISH reveals localization and copy number of Emi1 mRNA during cell division. RPE1 cell in which microtubules (red) and DNA (blue) are visualized. Individual molecules of Emi1 mRNA are stained using single molecule FISH (green).