Abstract
Receptor tyrosine kinase activity is typically measured by diverse biochemical methods detecting the amount of phosphorylation of proteins within a cell lysate. In this chapter, we present biophysical methods that allow for studying the activation process of single receptors, in particular the human epidermal growth factor receptor (EGFR) family, in live cells. We describe optical tracking of quantum dot (QD)-labeled single receptors using the total internal reflection fluorescence microscopy (TIRFM), and initial steps of data analysis to identify the time-dependent variation of single-receptor diffusion, which can be widely applied to studying activation of various cell surface receptors.
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References
Hynes NE, MacDonald G (2009) ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol 21:177–184
Kedrin D, Wyckoff J, Boimel PJ et al (2009) ERBB1 and ERBB2 have distinct functions in tumor cell invasion and intravasation. Clin Cancer Res 15:3733–3739
Yarden Y, Sliwkowski MX (2001) Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127–137
Albanell J, Baselga J (1999) The ErbB receptors as targets for breast cancer therapy. J Mammary Gland Biol 4:337–351
Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141:1117–1134
Chung I et al (2010) Spatial control of EGF receptor activation by reversible dimerization on living cells. Nature 464:783–787
Arkhipov A et al (2013) Architecture and membrane interactions of the EGF receptor. Cell 152:557–569
Zhang XW, Gureasko J, Shen K, Cole PA, Kuriyan J (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125:1137–1145
Bae JH, Schlessinger J (2010) Asymmetric tyrosine kinase arrangements in activation or autophosphorylation of receptor tyrosine kinases. Mol Cells 29:443–448
Alvarado D, Klein DE, Lemmon MA (2010) Structural basis for negative cooperativity in growth factor binding to an EGF receptor. Cell 142:568–579
Norris DJ, Efros AL, Rosen M, Bawendi MG (1996) Size dependence of exciton fine structure in CdSe quantum dots. Phys Rev B Condens Matter 53:16347–16354
Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, Nitschke R, Nann T (2008) Quantum dots versus organic dyes as fluorescent labels. Nat Methods 5:763–775
Jacobson K, Saxton M, Webb WW, Kusumi A, Sheetz MP (1994) Surface particle movements and membrane dynamics. FASEB J 8:152–152
Saxton MJ (1987) Lateral diffusion in an archipelago. The effect of mobile obstacles. Biophys J 52:989–997
Saxton MJ (1994) Single-particle tracking: models of directed transport. Biophys J 67:2110–2119
Saxton MJ (2004) A biological interpretation of anomalous subdiffusion. Biophys J 86:369A–369A
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Chung, I., Mellman, I. (2015). Single-Molecule Optical Methods Analyzing Receptor Tyrosine Kinase Activation in Living Cells. In: Germano, S. (eds) Receptor Tyrosine Kinases. Methods in Molecular Biology, vol 1233. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1789-1_4
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DOI: https://doi.org/10.1007/978-1-4939-1789-1_4
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