The investigation of mechanisms of action of endogenous, etiological or pharmacological agents can often be accelerated by acquisition of single-cell dynamic signalling data from cells responding to the exposure to such agents. High-throughput live-cell imaging methods are particularly sensitive because they allow large-scale comparison of individual cells before, during and after exposure to reagents. In particular response heterogeneity in a cell population, whether anticipated or not, can easily be identified and quantified.

Meanwhile, in drug discovery, secondary validation of specificity of protein interaction inhibitors may require negative screening for off-target effects, whereas it is necessary to demonstrate target engagement by novel reagent on the intended target in intact cells.

In all cases high-throughput imaging and related assays of cell signalling events are valuable tools. The unit is active in developing, implementing and validating an increasing range of cell signalling reporters, and packaging them in easy-to-use and safe gene-transfer vectors to facilitate these approaches. Contact us for more details.

Figure A shows stimulus-evoked signalling responses from an example of individual neighbouring cells. Figure B shows average responses from a field of synchonised cells under specific conditions. Figure C shows a automated specificity screen for off-target effects using compounds identified from a high-throughput screen and separately validated for target engagement, revealing one compound (#4) for elimination. Figure D shows an example subframe from a high-throughput migration screen.

Contact: Michael Courtney (michael.courtney [at] utu.fi)

References

1. Li LL, Ginet V, Liu X, Vergun O, Tuittila M, Mathieu M, Bonny C, Puyal J, Truttmann AC, Courtney MJ (2013) The nNOS-p38MAPK pathway is mediated by NOS1AP during neuronal death. J Neurosci. 33, 8185-8201. doi: 10.1523/JNEUROSCI.4578-12.2013 PMID: 23658158
2. Li LL, Melero-Fernandez de Mera RM, Chen J, Ba W, Nadif Kasri N, Zhang M, Courtney MJ (2015) Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models. J Neurosci. 35, 7349-7364. doi: 10.1523/JNEUROSCI.0037-15.2015 PMID: 25972165
3. Li LL, Cisek K, Courtney MJ (2017) Efficient Binding of the NOS1AP C-Terminus to the nNOS PDZ Pocket Requires the Concerted Action of the PDZ Ligand Motif, the Internal ExF Site and Structural Integrity of an Independent Element. Front. Mol. Neurosci. 10:58. doi: 10.3389/fnmol.2017.00058. PMID: 28360833.
4. Melero-Fernandez de Mera RM*, Li LL*, Popinigis A, Cisek K, Tuittila M, Yadav L, Serva A, Courtney MJ (2017) A simple optogenetic MAPK inhibitor design reveals resonance between transcription-regulating circuitry and temporally-encoded inputs. (*equal contribution) Nat. Commun. 8, 15017. doi: 10.1038/ncomms15017. PMID: 28497795.
5. Robinson S, Courtney MJ. Spatial quantification of the synaptic activity phenotype across large populations of neurons with Markov random fields. Bioinformatics. 2018 Apr 20. doi: 10.1093/bioinformatics/bty322. [Epub ahead of print] PubMed PMID: 29897415.
   

Optogenetics greatly simplifies the high-throughput application of a range of conditions in a more flexible way than liquid handling alone can offer. Our facility is experienced with integration of complex signalling pathway regulation paradigms using cellular optogenetic approaches (see reference below that revealed the existince of circuit resonance in mammalian MAPK pathways, as summarised in the figure). This approach can be combined with liquid handling if needed, or synchronised with automated image acquisition or other measurements. Contact us for more details.

Contact: Michael Courtney (michael.courtney [at] utu.fi)

References

Melero-Fernandez de Mera RM*, Li LL*, Popinigis A, Cisek K, Tuittila M, Yadav L, Serva A, Courtney MJ (2017) A simple optogenetic MAPK inhibitor design reveals resonance between transcription-regulating circuitry and temporally-encoded inputs. (*equal contribution) Nat. Commun. 8, 15017. doi: 10.1038/ncomms15017. PMID: 28497795.