Quantification of receptor tyrosine kinase transactivation through direct dimerization and surface density measurements in single cells

Abstract

Cell signaling involves dynamic changes in protein oligomerization leading to the formation of different signaling complexes and modulation of activity. Spatial intensity distribution analysis (SpIDA) is an image analysis method that can directly measure oligomerization and trafficking of endogenous proteins in single cells. Here, we show the use of SpIDA to quantify dimerization/ activation and surface transport of receptor protein kinases-EGF receptor and TrkB-at early stages of their transactivation by several G protein-coupled receptors (GPCRs). Transactivation occurred on the same timescale and was directly limited by GPCR activation but independent of G-protein coupling types. Early receptor protein kinase transactivation and internalization were not interdependent for all receptor pairs tested, revealing heterogeneity between groups of GPCRs. SpIDA also detected transactivation of TrkB by dopamine receptors in intact neurons. By allowing for time and space resolved quantification of protein populations with heterogeneous oligomeric states, SpIDA provides a unique approach to undertake single cell multivariate quantification of signaling processes involving changes in protein interactions, trafficking, and activity.high content analysis | oligomers | monoamine | N-acetyl cystein | brain-derived neurotrophic factor C ell surface receptor signaling involves dynamic spatial and temporal changes in protein oligomeric states, resulting in an intricate choreography of protein-protein interactions balanced with changes in receptor trafficking. For instance, activation of receptor tyrosine kinases (RTKs) by their cognate ligands enhances receptor dimer formation, internalization, and recruitment of monomeric receptors to the cell surface (1). Furthermore, RTKs can also be transactivated by G protein-coupled receptors (GPCRs) under certain circumstances (2-4). However, little is known about the dynamic effects of transactivation on RTK dimerization and surface trafficking. Indeed, the majority of research directed to understanding transactivation has been limited to biochemical assays (2-4) and provides limited information on the dynamics and/or spatial localization of this process within specific regions/compartments of cells/tissue.Understanding receptor dynamics requires methods that can be applied to cells or tissue under physiologically relevant, nonsteady state conditions. Spatial intensity distribution analysis (SpIDA) is a method based on the fitting of fluorescence intensity histograms calculated from conventional laser-scanning confocal images to monitor protein oligomerization in live or fixed cells and tissues (5). By directly monitoring early interactions between endogenous proteins in single cells rather than relying on the detection of downstream signaling molecules, SpIDA potentially offers several advantages over conventional assays used to study cell surface receptor distributions (5). We apply SpIDA and compare it with fluorescence lifetime imaging microscopy (FLIM) to extract information about t...

Revealing protein oligomerization and densities in situ using spatial intensity distribution analysis

“…Detection of dimers was confirmed with Förster resonance energy transfer (FRET) measurements in Swift et al (17). EGF-induced dimerization occurred even in the absence of clustering and internalization (14,17). The results show that SpIDA can resolve dynamic changes in receptor oligomerization within living cells, showing the power of the technique and its applicability to fluorescent protein expression systems.…”

“…Detection of dimers was confirmed with Förster resonance energy transfer (FRET) measurements in Swift et al (17). EGF-induced dimerization occurred even in the absence of clustering and internalization (14,17).…”

“…Direct comparisons of the two techniques are presented in ref. 17. SpIDA, nevertheless, presents several advantages.…”

Age-dependent deficits in fear learning in heterozygous BDNF knock-out mice

“…Several substances like, e.g., Zn ions (Hwang et al 2005), Cu ions (Hwang et al 2007), dopamine (Swift et al 2011), or adenosine (see, e.g., Tebano et al 2010) have been reported recently to transactivate Trk receptors. It thus seems possible that one or several of these transactivating factors of the TrkB-receptor undergo a decline during early post-puberty development in BDNF +/2 mice, thus causing the observed learning deficit in older BDNF +/2 mice.…”

The Spatiotemporal Organization of ErbB Receptors: Insights from Microscopy

“…Because this analysis does not depend on temporal correlation, it can extract data from a single image. Swift et al (2011) applied SpIDA to simultaneously monitor ErbB1 dimerization and internalization (concentration at the membrane) in response to transactivation via G-protein coupled receptors (GPCR). They found a differential response depending on the specific GPCR involved; all GPCR transactivation induced ErbB1 dimerization, yet not all of the GPCRs induced rapid receptor internalization.…”