Fluorescence Fluctuation Analysis of Receptor Kinase Dimerization
Receptor kinases are essential for the cellular perception of signals. The classical model for activation of the receptor kinase involves dimerization, induced by the binding of the ligand. The mechanisms by which plant receptors transduce signals across
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1. Introduction The somatic embryogenesis receptor kinase (SERK) was isolated as a marker for single cells in carrot (Daucus carota) cell suspension cultures that had acquired the ability to initiate somatic embryogenesis (1). SERK1 is a plasma membrane localized leucine-rich repeat (LRR)-receptor-like kinase (RLK) and consists of an N-terminal leucine zipper (LZ) domain, five LRRs, a proline-rich stromal processing peptidase (SPP) domain, a transmembrane domain, and an active intracellular serine/threonine kinase (2, 3). Förster Resonance Energy Transfer (FRET) studies have verified the interaction between SERK1 and BRI1-associated receptor kinase (BAK1)/SERK3, Brassinosteroid insensitive 1 (BRI1) and other proteins in confined regions at the plasma membrane (4, 5). To study BRI1, SERK1, and SERK3 by
N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_11, © Springer Science+Business Media, LLC 2011
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fluorescence techniques its cDNA has been C-terminally fused to the cyan (CFP) or yellow (YFP) variant of enhanced green fluorescent protein (EGFP) (3) and transiently expressed in plant cells. Both BRI1 and SERK1 receptors were shown to be present in an oligomerized state in the plasma membrane (3). Elimination of the extracellular LZ domain reduced the FRET efficiency to control levels, indicating that SERK1 lacking the LZ domain (SERK1∆LZ) is monomeric (3). However, standard imaging techniques to visualize FRET with high-spatial resolution such as confocal microscopy (6) and fluorescence lifetime imaging microscopy (FLIM) (7) require a relatively large amount of fluorescently tagged molecules (>1 mM). This may be well above physiologically relevant concentrations. In addition, it is difficult to retrieve information about the oligomeric status of the studied proteins. Techniques such as photon counting histogram (PCH) analysis (8) and fluorescence correlation spectroscopy (FCS) (9) can provide this information and operate at the single-molecule detection level, allowing to measure at physiologically relevant concentrations instead of using overexpression conditions. For FCS and PCH, the fluorescence intensity is monitored in a small observation volume that is continuously illuminated. A particle with a given molecular brightness, h, produces an intensity fluctuation as it passes the observation volume. Particles with a higher molecular brightness will result in stronger intensity fluctuations. Since small particles will diffuse more rapidly through the observation volume than large molecules, the duration of the fluorescence bursts contains information on the diffusion speed of the particles. PCH and FCS use the same experimental data, but each technique focuses on a different property of the signal. FCS analyses the time-dependent decay of the fluorescence fluctuations, while PCH calculates the amplitude distribution of these fluctuations. This latter technique yields the distribution of mole
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