Automated nanoscale flow cytometry for assessing protein–protein interactions

Abstract

Despite their importance for signalling events, protein-protein interactions cannot easily be analyzed on a single cell level. We developed a robust automated FRET measurement system implemented on a commercial flow cytometer allowing for rapid profiling of molecular associations in living cells. We used this method to measure the most proximal signaling events on human T lymphocyte activation, which preceded calcium influx, and could automatically detect T cell receptor/CD3 complex clustering defects in immunocompromised patients. V C 2016 International Society for Advancement of Cytometry Key terms flow cytometry; FRET; T cells; automatic data analysis; detection of immunodeficiencies F LOW cytometry is commonly used to detect and quantify overall fluorescent signals associated with cells. While this method can give detailed quantitative and multimodal information on the presence of target molecules, no information on the distribution or interaction between molecules of interest can be gained from these signals. Yet, protein-protein interactions govern most aspects of biological functions. For example, during the activation process of a human T lymphocyte by an antigen presenting cell, the T cell receptor (TCR)/CD3 receptor complex is known to cluster with the CD4 coreceptor (1) on the cell surface of CD4-positive cells. Additionally, homoclustering of the TCR/CD3 complex within the central immunological synapse can be observed and is critical for signal initiation (2).F€ orster resonance energy transfer (FRET) is a radiation-free process in which energy from a donor molecule in the excited state is transferred to an acceptor molecule via dipole-dipole interactions (3). FRET is commonly used as a spectroscopic ruler (4) as the energy transfer can only occur for distances up to 10 nm. The effect can be measured via a decrease in the fluorescence intensity of FRET donor molecules and a concomitant increase in the intensity of acceptor molecules (when excited at the donor wavelength) (5). Molecular interactions usually take place in the range of up to 10 nm, and therefore FRET is an excellent tool to detect molecular proximities.There are still some major limitations in the measurement of FRET. Traditionally, FRET signals were measured by microscopy, which produces large amount of data and requires much expert knowledge for data analysis. To facilitate the complex microscopic FRET analysis, several programs that simplify the microscopic intensitybased FRET analysis were developed (6). Flow cytometric FRET analysis offers better statistical information (5) on the FRET efficiency (E) and thus can be used as a high throughput technique to discover molecular associations. Yet, because of the spectral overlap between donor and acceptor fluorescence (necessary for any FRET measurement), the spillover in the regarding channels could eliminate a clear FRET signal. Therefore, numerous controls have to be included in the experiment and complex

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“…Secretory granules in alpha cells have been previously studied using transmission electron microscopy and their average sizes have been reported to be in the range of 180–240 nm (29–31). Accordingly, we confirmed the presence of secretory granules using nano-scale flow cytometry with Fc-glucagon as an exclusive marker for alpha cell secretory granules (32, 33). We used beads in the range of 110–880 nm for calibration in the range of the reported sizes for secretory granules (Supplementary Figure 2A).…”

The flow of events: How the sequence of molecular interactions is seen by the latest, user‐friendly high throughput flow cytometric FRET

“…The "on-machine", rapid FRET assay described in the article by von Kolontaj et al (11) completely fulfills this criterion. The "on-machine", rapid FRET assay described in the article by von Kolontaj et al (11) completely fulfills this criterion.…”

“…The introduction of an easy to use flow cytometric FRET method capable of high throughput analysis would definitely further boost the number of publications in the FRET field. The "on-machine", rapid FRET assay described in the article by von Kolontaj et al (11) completely fulfills this criterion. The authors describe a fully automated nanoscale flow cytometry assay to detect protein-protein interactions in a commercial flow cytometer.…”

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