Bumpus, Timothy William2021-03-152023-01-112020-12Bumpus_cornellgrad_0058F_12303http://dissertations.umi.com/cornellgrad:12303https://hdl.handle.net/1813/103364169 pagesChemical imaging techniques have been instrumental in advancing our understanding of the spatiotemporal regulation of signal transduction pathways. Phospholipase D (PLD) enzymes are a critical signaling node responsible for production of the pleiotropic lipid second messenger phosphatidic acid via hydrolysis of phosphatidylcholine. It remains a mystery how this one lipid signal can cause such diverse physiological and pathological signaling outcomes, due in large part to a lack of suitable tools for visualizing the spatial and temporal dynamics of its production within cells. Here, we report a chemical method for imaging and quantifying PLD activity in live cells. Our approach, termed IMPACT (Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation), capitalizes upon the enzymatic promiscuity of PLDs, which we show can accept a wide variety of chemically functionalized alcohols as reporters of their activity in a transphosphatidylation reaction. We can then fluorescently tag the resultant phosphatidyl alcohol lipids using click chemistry, enabling us to visualize both cellular membranes bearing active PLD enzymes and quantify cellular PLD activity across a population of labeled cells, because the level of labeling directly correlates with enzymatic activity. Our method specifically reveals the subcellular sites of PLD activity and allows us to sort a population of cells as a function of their PLD activity. The single cell resolution of IMPACT quantification proved instrumental in its utility as a readout for a genome wide pooled CRISPR interference screen to identify regulators of protein kinase C (PKC)-dependent PLD signaling. Combining these two modern chemical tools has proven to be extremely powerful and allowed us to identify a new role for glycogen synthase kinase 3 (GSK3) in regulating the transcription of PKC and PLD. This finding reveals that PKC levels are self-regulating as high PKC activity inhibits GSK3 and thus reduces its own transcription. Collectively, our studies developing and utilizing IMPACT highlight the importance and power of using chemical tools to directly visualize the activity of cellular signaling enzymes.endissertation or thesishttps://doi.org/10.7298/g2za-jm73