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Item A novel micellular fluorogenic substrate for quantitating the activity of 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma (PLCγ) enzymes(Public Library of Science, 2024-03-29) Visvanathan, Ramya; Utsuki, Tadanobu; Beck, Daniel E.; Clayton, W. Brent; Lendy, Emma; Sun, Kuai-lin; Liu, Yinghui; Hering, Kirk W.; Mesecar, Andrew; Zhang, Zhong-Yin; Putt, Karson S.; Pharmacology and Toxicology, School of MedicineThe activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes.Item Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases(Royal Society of Chemistry, 2020) Jena, Sampreeti; Damayanti, Nur P.; Tan, Jackie; Pratt, Erica D.; Irudayaraj, Joseph M.K.; Parker, Laura L.; Neurological Surgery, School of MedicineMany commonly employed strategies to map kinase activities in live cells require expression of genetically encoded proteins (e.g. FRET sensors). In this work, we describe the development and preliminary application of a set of cell-penetrating, fluorophore labelled peptide substrates for fluorescence lifetime imaging (FLIM) of Abl and Src-family kinase activities. These probes do not rely on FRET pairs or genetically-encoded protein expression. We further demonstrate probe multiplexing and pixel-by-pixel quantification to estimate the relative proportion of modified probe, suggesting that this strategy will be useful for detailed mapping of single cell and subcellular dynamics of multiple kinases concurrently in live cells.Item PIE-FLIM Measurements of Two Different FRETBased Biosensor Activities in the Same Living Cells(Cell Press, 2020-04-21) Reissaus, Christopher A.; Day, Kathleen H.; Mirmira, Raghavendra G.; Dunn, Kenneth W.; Pavalko, Fredrick M.; Day, Richard N.; Pediatrics, School of MedicineWe report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.