Summary
How a eukaryotic cell translates a small concentration difference of a chemoattractant across the length of its surface into highly polarized intracellular responses is a fundamental question in chemotaxis. Chemoattractants are detected by G-protein-coupled receptors (GPCRs). Binding of chemoattractants to GPCRs induces the dissociation of heterotrimeric G-proteins into Gα and Gβγ subunits, which in turn, activate downstream signaling networks. To fully understand the molecular mechanisms of chemotaxis, it is essential to quantitatively measure the dynamic changes of chemoattractant concentrations around cells, activation of heterotrimeric G-proteins, and the mobility of GPCR and G-protein subunits in the cell membrane. Here, we outline fluorescence imaging methods including Förster resonance energy transfer (FRET) imaging and a single-molecule analysis that allow us to measure the dynamic properties of GPCR signaling in single live cells.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Moser, B. and Loetscher, P. (2001) Lymphocyte traffic control by chemokines. Nat. Immunol. 2, 123–128.
Murphy, P. M. (2002) International Union of Pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacol. Rev. 54, 227–229.
Jin, T., Xu, X., and Hereld, D. (2008) Chemotaxis, chemokine receptors and human disease. Cytokine 44, 1–8.
Devreotes, P. N. and Zigmond, S. H. (1988) Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium. Annu. Rev. Cell Biol. 4, 649–686.
Jin, T. and Hereld, D. (2006) Moving toward understanding eukaryotic chemotaxis. Eur. J. Cell Biol. 85, 905–913.
Fang, J., Brzostowski, J. A., Ou, S., Isik, N., Nair, V., and Jin, T. (2007) A vesicle surface tyrosine kinase regulates phagosome maturation. J. Cell Biol. 178, 411–423.
Devreotes, P. N. (1994) G protein-linked signaling pathways control the developmental program of Dictyostelium. Neuron 12, 235–241.
Chung, C. Y., Funamoto, S., and Firtel, R. A. (2001) Signaling pathways controlling cell polarity and chemotaxis. Trends Biochem. Sci. 26, 557–566.
Iijima, M., Huang, Y. E., and Devreotes, P. (2002) Temporal and spatial regulation of chemotaxis. Dev. Cell 3, 469–478.
Xu, X., Meier-Schellersheim, M., Jiao, X., Nelson, L. E., and Jin, T. (2005) Quantitative imaging of single live cells reveals spatiotemporal dynamics of multistep signaling events of chemoattractant gradient sensing in Dictyostelium. Mol. Biol. Cell 16, 676–688.
Xu, X., Meier-Schellersheim, M., Yan, J., and Jin, T. (2007) Locally controlled inhibitory mechanisms are involved in eukaryotic GPCR-mediated chemosensing. J. Cell Biol. 178, 141–153.
Xu, X., Brzostowski, J. A., and Jin, T. (2006) Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells. Methods Mol. Biol. 346, 281–296.
Isik, N., Brzostowski, J. A., and Jin, T. (2008) An Elmo-like protein associated with myosin II restricts spurious F-actin events to coordinate phagocytosis and chemotaxis. Dev. Cell 15, 590–602.
Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.
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.
Acknowledgments
The work is supported by Intramural Research Program of the NIH/NIAID and the NIH Intramural AIDS Targeted Antiviral Program.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press
About this protocol
Cite this protocol
Xu, X., Brzostowski, J.A., Jin, T. (2009). Monitoring Dynamic GPCR Signaling Events Using Fluorescence Microscopy, FRET Imaging, and Single-Molecule Imaging. In: Jin, T., Hereld, D. (eds) Chemotaxis. Methods in Molecular Biology™, vol 571. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-198-1_25
Download citation
DOI: https://doi.org/10.1007/978-1-60761-198-1_25
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-197-4
Online ISBN: 978-1-60761-198-1
eBook Packages: Springer Protocols