- Browse by Subject
Browsing by Subject "Fluorescence resonance energy transfer"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Direct visualization by FRET-FLIM of a putative mechanosome complex involving Src, Pyk2 and MBD2 in living MLO-Y4 cells(PLOS, 2021-12-23) Day, Richard N.; Day, Kathleen H.; Pavalko, Fredrick M.; Anatomy, Cell Biology and Physiology, School of MedicineEarlier, we proposed the "mechanosome" concept as a testable model for understanding how mechanical stimuli detected by cell surface adhesion molecules are transmitted to modulate gene expression inside cells. Here, for the first time we document a putative mechanosome involving Src, Pyk2 and MBD2 in MLO-Y4 osteocytes with high spatial resolution using FRET-FLIM. Src-Pyk2 complexes were concentrated at the periphery of focal adhesions and the peri-nuclear region. Pyk2-MBD2 complexes were located primarily in the nucleus and peri-nuclear region. Lifetime measurements indicated that Src and MBD2 did not interact directly. Finally, mechanical stimulation by fluid flow induced apparent accumulation of Src-Pyk2 protein complexes in the peri-nuclear/nuclear region, consistent with the proposed behavior of a mechanosome in response to a mechanical stimulus.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.