Multiplexed Spatial Analyses in situ and in living cells
Diehl, Michael R
Doctor of Philosophy
High-content spatial analyses are critical to understanding the structural organization and dynamics of many complex biological processes. Increasing the number of cellular components that can be visualized will help delineate the functions of many interacting and competing cellular pathways. However, the physical limitations of spectral bandwidth and the experimental difficulty of genomic manipulation have hampered traditional approaches to multiplex molecular analyses in both fixed samples and live cells. The programmable and predictable nature of the DNA molecule makes it a tantalizing candidate for an engineering tool to help alleviate some of these limitations. This thesis seeks to harness both the chemical and biological utility of DNA as a building block to multiplex the color and control the number and location of fluorescent reporters in biological samples. First in the context of in situ immunofluorescence imaging of fixed cells or tissues, And second in the context of live-cell imaging of genomically engineered cells. In the first case, by utilizing the stand displacement chemical reaction between dynamic DNA complexes and DNA-conjugated antibodies we selectively couple fluorophores to, and then remove them, from their protein targets. We leverage this mechanism to facilitate multiple sequential round of fluorescence microscopy where the same color dye molecules are used reiteratively to visualize different antibody-tagged markers. By optimizing the DNA-antibody conjugation chemistry and incubation protocol we now routinely perform 9 marker analyses of paraffin-fixed tissue sections with these DNA probes. Then automating the sequence design process enabled more complex probe designs to be use for balancing marker levels appropriately for hyperspectral imaging experiments. Here, discrete and reconfigurable control over amplification gains, greatly improved the spectral un-mixing of different antibody signals. Secondly, we focus on dissecting network-level functions of cytoskeletal regulatory proteins during epithelial cell polarization and morphogenesis. DNA-based STORM microscopy revealed that a scaffold protein, IQGAP1, associates with specialized actin filaments within cell-cell junctions and with basket-like structures in the basal actin cortex of normal epithelial cells. This work uses IQGAP1 as a platform, as it lies at the nexus of cell signaling and cytoskeletal regulatory networks. We construct multi-gene systems that simultaneously sense and control intracellular expression levels of IQGAP1 and track the actin cytoskeleton. By combining novel molecular biology techniques to manipulate the DNA in live cells. We use a barcoded self-assembly technique to construct large vectors that contain several transcriptional elements. These multi-gene systems are then stably incorporated into cells engineered with genomic ‘landing pads’ using locus-specific integration. Finally, we demonstrate functional circuits by linearly controlling intracellular IQGAP1 levels. These results will support future single-cell and multiplexed population-level analyses of IQGAP1 functions in epithelial cells, allow us to study IQGAP1 recruitment to epithelial cell-cell junctions and to examine how it influences cellular transitions.
DNA Nanontechnology; Immunofluorescence; Synthetic Biology