Fluorescence Correlation Spectroscopy and Fluorescence Recovery After Photobleaching to Study Receptor Kinase Mobility I
Plasma-membrane-localized receptor kinases are essential for cell–cell communication and as sensors for the extracellular environment. Receptor function is dependent on their distribution in the membrane and interaction with other proteins that are either
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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 SPP domain, a transmembrane domain, and an active intracellular serine/threonine kinase (2, 3).
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_13, © Springer Science+Business Media, LLC 2011
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At the cellular level, Förster Resonance Energy Transfer (FRET)-based techniques have verified the interaction between SERK1 and 14-3-3 (4), CDC48 (5), KAPP (6), SERK2 (7), BRI1 and BAK1/SERK3 (5). These studies showed that the interaction between SERK1 and its partners is highly dynamic. As reported for BRI1 and BAK1/SERK3 (8), SERK1 also interacts with SERK2, BAK1/SERK3, BRI1, and CDC48 in confined regions at the plasma membrane (5, 7, 9, 10) and with KAPP only after internalization (6). For BRI1 and BAK1/SERK3, it was postulated that the interaction occurs just prior to endocytosis (8). Both BRI1 and SERK1 receptors are also present in a homodimerized state in the plasma membrane (3, 8, 9, 11). SERK1 homodimerization depends on the presence of the extracellular LZ domain (3). Photon counting histogram analysis and fluorescence cross-correlation microscopy in plant protoplasts showed that 13% of the SERK1 receptor and 20% of the BRI1 receptor proteins are present in a homodimerized state. BRI1 homodimerization in protoplasts seems independent of the presence of brassinosteroids (12). Mass spectrometry-based data suggest that BRI1 activation results in increased interaction with the BAK1/SERK3 coreceptor (13). A Fluorescence Recovery After Photobleaching (FRAP) mobility study of FLS2, the plant receptor for flagellin, showed in protoplasts that both the FLS2 mobile fraction and diffusion coefficient are reduced in response to its ligand (14). Fluorescence Fluctuation Spectroscopy (FFS)-based techniques enable the measurement of protein dynamics and interactions in living cells at physiological protein concentrations. FFS utilizes a confocal microscope with high numerical aperture (NA) objective that reduces the observation volume to less than 1 fL (15, 16). This also enables measurements at the single molecule level using sensitive avalanche photodiode detectors. Since these techniques are noninvasive, they are very attractive tools for use in living cells (16–18). In an FFS experiment, spontaneous fluorescence fluctuations around an equilibrium caused by continuously excited single molecules diffusing into and out of the observation volume are observed (15, 19). Fluorescence Correlation Spectroscopy (FCS) studies the temporal behavior of fluctuations in fluorescence intensity and is analyzed by calcula
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