by Dr C. J. Caunt, S. P. Armstrong and Prof. C. A. McArdle
Established analytical techniques for cellular research are typically destructive and time-consuming. They limit the number and complexity of assays that can be performed and make it difficult to understand the true biological context of the results. Conversely, high-content analysis employs image based cellular assays in a high-throughput imaging and analysis format. This allows researchers to increase the number of questions they can ask whilst simultaneously decreasing the time taken to achieve their results.

Vast numbers of extracellular signalling molecules influence target cells by activating a relatively limited number of mitogen-activated protein kinase (MAPK) cascades. Specificity largely depends on differences in kinetics and localisation of MAPK activation; these differences are in turn dictated by MAPK associated proteins serving as scaffolds, anchors, activators or effectors. Elucidating the various parameters influencing signalling will play a vital role in describing both healthy and disease states and identifying appropriate therapeutic targets.

The Ras/Raf extracellular signal regulated kinase (ERK) transduction cascade plays a critical role in cellular profileration, making it an important target for cancer studies. We monitored this pathway closely and explored the spatial and temporal aspects of signalling via the Ras/Raf ERK pathway by G protein-coupled receptors and tyrosine kinase receptors [1–3].  

We were particularly interested in the molecular mechanisms underlying these differences but the time-consuming techniques used to monitor ERK activation imposed major bottlenecks on our studies. We therefore evaluated high-content analysis (HCA) using the IN Cell Analyzer 1000 (GE Healthcare Life Sciences), and this led to the development of an efficient means of monitoring spatial and temporal aspects of ERK signalling based on siRNA knock-down of endogenous ERKs, adenovirus (Ad)-mediated add-back of wild-type, or modified ERK2-GFP reporters [4].

HIGHER THROUGHPUT
Figure 1 shows images of ppERK1/2 staining in HeLa cells stimulated with either EGF or the PKC-activator PDBu in the presence or absence of AG1478, an inhibitor of EGF receptor activation, and RO-318425, a PKC inhibitor. The effect of the stimuli and inhibitors is immediately evident from the thumbnails, and quantitation of whole cell ppERK1/2 staining yields dose- and time-dependencies similar to those previously obtained by Western blotting (data not shown). However, the HCA methodology used in conjunction with IN Cell Analyzer 1000 resulted in much higher throughput. Image acquisition for the entire 96-well plate was completed within 20 min and image analysis (over 50 000 individual cells) took a comparable amount of time. For comparison, preparation of both nuclear and cytoplasmic extracts for Western blotting (two antibodies and 96 samples) would have taken at least several days and typically weeks to complete.

BETTER INSIGHTS
IN Cell Analyzer 1000 also provided additional detail on subcellular compartmentalisation of ERK within cells. Scatterplots tracking ERK activation in control cells and cells stimulated with EGF or PDBu show that both factors increase nuclear and cytoplasmic ppERK [figure 2]. They also reveal that PDBu is more efficient at causing ppERK translocation to the nucleus. This information is critically important to the mechanics of this pathway, but it could not have been obtained by Western blotting with whole cell extracts or by an ELISA-type ppERK assay.

INNOVATIVE MONITORING
Since the phosphorylation state of ERK influences binding to some (but not all) ERK scaffolds, we wanted to be able to monitor subcellular distribution of ERK and ppERK in parallel. First, we knocked down expression of endogenous ERKs 1 and 2 using siRNAs (targeting noncoding regions). Then we used recombinant adenovirus (Ad) to add back specific ERK expression (either Ad-mediated ERK2/GFP, which is functionally indistinguishable from ERK2 and can be expressed at physiological levels using appropriate Ad titre) or Y261A and D319N mutants of ERK2/GFP (mutants that have impaired binding to the DEF or D-domains typically favoured by ERK. The efficiency of the knock-down/add-back strategy and the effect of EGF on activation and translocation of the wild-type and mutated ERK2/GFP reporters is illustrated in figure 3.

We deployed the same knock-down/add-back monitoring method to elucidate the stimulus-specific effects of dual-specificity phosphatases (DUSPs) on ERK signalling. DUSPs are one of the most important scaffolds and regulators of ERK signalling and have been investigated as potential therapeutic targets. We used the knock-down/add-back protocol to screen an siRNA library targeting functional MAPK phosphatases as well as 10 structurally related atypical DUSPs to determine acute and chronic responses (ppERK elevation and ERK2-GFP translocation) to EGF and PDBu. Sixteen of the 20 DUSPs screened (including atypical DUSPs) influenced ERK signalling [figure 4], and the effects seen were dependent on the stimulus and end-point measured (data not shown). This specificity (e.g. the lack of functional redundancy within this protein family) supports their potential value as therapeutic targets.

LIVE-CELL IMAGING
Translocation biosensors can also be used for live-cell imaging with the IN Cell Analyzer 1000 Environmental Control Module and this can provide valuable information on the kinetics of biological responses. We have used the ERK knock-down/add-back method to test for desensitisation of ERK responses during a series of brief stimuli. As shown [figure 5], when GPCRs expressed in HeLa cells received four brief periods of stimulation (5 min stimulation followed by 55 min in control medium), each stimulus elicited a rapid and transient translocation of ERK2-GFP to the nucleus (indicating ERK activation) but there was no obvious desensitisation (e.g. the fourth response was comparable to the first).

CONCLUSION
Semi-automated image acquisition and analysis with HCA using the IN Cell Analyzer 1000 provides an extremely efficient means of monitoring ERK signalling. The high-throughput achieved with this approach has facilitated development of a knock-down/add-back protocol for interrogation of spatio-temporal aspects of responses influenced by ERK mutations and phosphatase knock-down. Surprisingly, many DUSPs influenced ERK signalling and did so in different ways (e.g., selectively influencing acute or chronic responses, or selectively influencing response to EGF and PDBu). This argues strongly against functional redundancy; a point that will be relevant in the exploitation of DUSPs as therapeutic targets. Importantly, the wide range of effects discovered would not have been evident in a single end-point assay (e.g., Western blotting or ELISA for ppERK). The high-content approach afforded by IN Cell Analyzer 1000 not only increases throughput and time savings, but also leads to better data and more knowledge about cellular systems.

REFERENCES
1. Caunt CJ et al. Arrestin-mediated ERK activation by gonadotropin-releasing hormone receptors (GnRHRs): Receptor-specific activation mechanisms and compartmentalization. J Biol Chem 2006; 281: 2701–2710.
2. Caunt CJ et al. GnRH Receptor Signaling to ERK: Kinetics and Compartmentalization. Trends in Endocrinology and Metabolism 2006; 17: 76–283.
3. Caunt CJ et al. Seven Transmembrane Receptor Signaling and ERK Compartmentalization. Trends in Endocrinology and Metabolism 2006; 17: 308–313.
4. Caunt CJ et al. EGF receptor and protein kinase C signaling to ERK2: Spatiotemporal regulation of ERK2 by dual-specificity phosphatases. J Biol Chem 2008; 283: 6241-52.

THE AUTHORS
Dr C. J. Caunt, S.P. Armstrong and Prof. C. A. McArdle,
Labs for Integrative neuroscience and Endocrinology,
Dept of Clinical Sciences at South Bristol,
University of Bristol,
UK