Cross Correlation of Transcription Factor Binding and RNA Synthesis in Saccharomyces cerevisiae by 3D Orbital Tracking

This project utilized 3D orbital tracking and a newly developed fluorescent labeling strategy that allows simultaneous visualization of pre-mRNA and transcription factors inside of living yeast cells. This allowed us to follow the fate of individual eukaryotic pre-mRNA molecules as they undergo transcription in real time and enable complete kinetic characterization of the initiation, elongation and release of individual RNA molecules as well as single molecule temporal correlation of transcription factor binding to DNA. In previous work, due to the rapid photobleaching of cells, 10-20 measurements were averaged together to determine transcriptional kinetics. With 3D orbital tracking, the information garnered in three previous experiments on two separate microscopes will be available in a single cell measurement at a 100x times faster sampling rate

Deconstructing the Cell’s Mechanical Circuits by 3D Orbital Tracking Microrheology

We seek to address a critical question in physical cell biology: is it possible to develop an ‘inventory’ of mechanical or force-sensing modules of cell and tissue behavior in analogy to the modules in biochemical signaling networks? To discover these mechanical modules we focus on a specific system of broad interest - cell extrusion in epithelial sheets. We will measure how chemical perturbations affect the mechanics and rheology of both the cytoplasm and the extracellular matrix of living cells. To measure intracellular viscoelasticity we will track a fluorescent particle or organelle using high-resolution 3D orbital tracking and high speed video microscopy. The mean squared displacement of the particle vs. time provides a measure of the frequency-dependent complex viscoelastic modulus. Finally we will monitor the regression of vasculature during pathway inhibition to deconstruct the chemical-mechanical circuits that regulate the vessel growth and retraction known as anoikis

A Study of Transcriptional Activation by the Transcription Factor Gal4 in Saccharomyces cerevisiae by 3D Orbital Tracking and In Vivo RNA labelling

Understanding what is going on at the molecular level within individual cells is challenging. But deciphering these stochastic biomolecular processes is crucial for our understanding of gene transcription and the intricacies of cellular metabolism. With 3D orbital tracking we are able to visualize and monitor pre-mRNA and transcription factors in real-time using fluorescent tagging within yeast cells at a high sampling rate. Our study demonstrates that we can track these molecules of interest, fluorescent-labeled GFP (pre-mRNA) and JF 646 dye (transcription factors), during the process of transcribing a gene that codes for metabolizing galactose. This method allows us to directly observe the movement of a single molecule and determine the time lag between the GAL4 transcription factor binding to DNA and the activation of the mRNA synthesis. The data we collected improves our knowledge of the details of transcriptional kinetics and how single celled eukaryotic organisms regulate transcription. This will expand our research on the transcription processes in similar genes and in multicellular organisms like humans. 3D orbital tracking opens up a new window for exploring fundamental biochemical processes through a dynamic view of single fluorescent molecules in living systems at high speed. Coulon, A. et al., 2014. Kinetic competition during the transcription cycle results in stochastic RNA processing. eLife, 3. Available at: http://www.ncbi.nlm.nih.gov/pubmed/25271374

A Study of Transcriptional Activation by the Transcription Factor Gal4 in Saccharomyces Cerevisiae by 3D Orbital Tracking and in Vivo RNA Labelling

Understanding what is going on at the molecular level within individual cells is challenging. But deciphering stochastic biomolecular processes is crucial for our understanding of gene transcription and the intricacies of cellular metabolism. With 3D orbital tracking, we are able to visualize and monitor pre-mRNA and transcription factors in real-time using fluorescent tagging within yeast cells at a high sampling rate. Our study demonstrates that we can track these molecules of interest, fluorescent-labeled GFP (pre-mRNA) and JF 646 dye (transcription factors), during the process of transcribing a gene that codes for metabolizing galactose. This method allows us to directly observe the movement of a single molecule and determine the time lag between the GAL4 transcription factor binding to DNA and the activation of the mRNA synthesis. The data we collected improves our knowledge of the details of transcriptional kinetics and how single celled eukaryotic organisms regulate transcription. This will expand our research on the transcription processes in similar genes and in multicellular organisms like humans. 3D orbital tracking opens up a new window for exploring fundamental biochemical processes through a dynamic view of single fluorescent molecules in living systems at high speed