Abstract
The mechanisms that mediate how migratory eukaryotic cells amplify a shallow, extracellular chemoattractant gradient into a steep intracellular gradient of signaling components to guide chemotaxis remains unknown. To unravel these mechanisms, it is essential to quantitatively measure the spatiotemporal patterns of chemoattractant gradients, the dynamic movement of intracellular signaling pathway molecules, and the localized activation of these molecules in single living cells. Recent developments in live-cell fluorescence microscopy have permitted direct visualization and quantitative measurement of signal transduction events with high temporal and spatial resolution. Here, we outline fluorescence imaging methods to simultaneously visualize and quantitatively measure spatiotemporal changes in chemoattractant concentration by using the fluorescent tracer dye Alexa 594. Next, we provide a method to correlate the dynamic changes in ligand to the spatiotemporal changes in the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3) along the inner surface of the plasma membrane in live cells. Finally, we describe a fluorescence resonance energy transfer (FRET) method to determine the extent of heterotrimeric G protein activation in single living cells in response to various chemoattractant fields.
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Parent, C. and Devreotes, P. N. (1999) A cell’s sense of direction. Science 284, 765–770.
Devreotes, P. N. and Janetopoulos, C. (2003) Eukaryotic Chemotaxis: Distinctions between directional sensing and polarization. J. Biol. Chem. 278, 20,445–20,448.
Servant, G., Weiner, O. D., Herzmark, P., Balla, T., Sedat, J. W., and Bourne, H. R. (2000) Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 287, 1037–1040.
Zigmond, S. H., Levitsky, H. I., and Kreel, B. J. (1981) Cell polarity: an examination of its behavioral expression and its consequences for polymorphonuclear leukocyte chemotaxis. J. Cell Biol. 89, 585–592.
Xiao, Z., Zhang, N., Murphy, D. B., and Devreotes, P. N. (1997) Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation. J. Cell Biol. 139, 365–374.
Jin, T., Zhang, N., Long, Y., Parent, C. A., and Devreotes, P. N. (2000) Localization of the G protein βg complex in living cells during chemotaxis. Science 287, 1034–1036.
Iijima, M. and Devreotes, P. N. (2002) Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell 109, 599–610.
Funamoto, S., Meili, R., Lee, S., Parry, L., and Firtel, R. A. (2002). Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell 109, 611–623.
Parent, C., Blacklock, B., Froelich, W., Murphy, D., and Devreotes, P. N. (1998) G protein signaling events are activated at the leading edge of chemotactic cells. Cell 95, 81–91.
Xu, X., Meier-Schellersheim, M., Jiao, X., Nelson, L. E., and Jin, T. (2005) Quantitative imaging of single live cells reveals spatiotemporal dynamics of multi-step signaling events of chemoattractant gradient sensing in Dictyostelium. Mol. Biol. Cell 16, 676–688
Miyawaki, A. (2003) Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295–305
Janetopoulos, C., Jin, T., and Devreotes, P. N. (2001) Receptor mediated activation of heterotrimeric G-proteins in living cells. Science 291, 2408–2411.
Sekar, R. B. and Periasamy, A. (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localization. J. Cell Biol. 160, 629–633.
Jiao, X., Zhang, N., Xu, X., Oppenheim, J. J., and Jin, T. (2005) Ligand-induced partitioning of human CXCR1 chemokine receptors with lipid-raft microenvironments facilitates G-protein-dependent signaling. Mol. Cell. Biol. 25, 5752–5762.
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© 2006 Humana Press Inc., Totowa, NJ
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Xu, X., Brzostowski, J.A., Jin, T. (2006). Using Quantitative Fluorescence Microscopy and FRET Imaging to Measure Spatiotemporal Signaling Events in Single Living Cells. In: Eichinger, L., Rivero, F. (eds) Dictyostelium discoideum Protocols. Methods in Molecular Biology™, vol 346. Humana Press. https://doi.org/10.1385/1-59745-144-4:281
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DOI: https://doi.org/10.1385/1-59745-144-4:281
Publisher Name: Humana Press
Print ISBN: 978-1-58829-623-8
Online ISBN: 978-1-59745-144-4
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