11. Conclusions and future perspectives
Many microscopy and spectroscopy approaches are available for studies of GPCR signalling at multiple levels from individual molecules to whole organisms. Future developments in microscopy techniques, particularly SRN approaches (MINFLUX, RASTMIN, MINSTED, and RESI), will bridge the gap between optical microscopy and structural biology. More extensive use of the traditionally “in vitro ” techniques with high temporal resolution, such as smFRET and nanosecond FCS, in living cells and organisms will enable dynamic signalling studies in a more complex native setting. The utilisation of previously underappreciated optical phenomena and the development of label-free technologies will allow the extraction of additional information without increasing the complexity of experimental systems. These advances will allow the determination of structural and dynamic properties of GPCR signalling in conditions closer to natural than previously possible.
The development of new labelling approaches and small specific labels with enhanced optical properties will allow use of the full potential of new microscopy techniques. The use of CRISPR gene editing along with genetic code modification and unnatural amino acids will allow the targeted labelling of endogenous proteins with minimal perturbation to endogenous systems. The development of new small and specific labels, including high-affinity fluorescent ligands, dyes suitable for click-chemistry with unnatural amino acids, and ultimately fluorescent amino acids, will enable the precise detection of conformational changes, the localisation of cascade members, and their interactions with the environment and other proteins at the level of individual molecules in endogenous systems.
Machine learning and artificial intelligence (AI) are already revolutionising many fields of science, including microscopy, and signalling studies. Machine learning AI will enable a high degree of automatization in image acquisition and analysis, as well as the extraction of previously unavailable information from imaging data. It will likely lead to the development of new imaging approaches and revolutionise data analysis. The future conversion of complex microscopy approaches such as SPT into high-throughput techniques will qualitatively change the amount and accuracy of the information we can obtain to understand the properties of the GPCR signalling cascade.
In summary, future developments in microscopy, spectroscopy techniques and data analysis algorithms will unify the data obtained at multiple scales and allow researchers to understand endogenous signalling in unpreceded detail and precisely modulate its function.
Author contributions: All authors contributed to the conceptualisation, design, writing, editing of the manuscript and preparation of figures and tables.
Conflict of interest: MM is the Chief Scientific Officer of Celtarys Research S.L. TF and AB declare no potential conflict of interest.
Acknowledgements: The authors gratefully acknowledge the support from the COST Action CA18133 European Research Network on Signal Transduction (ERNEST). This work was supported by the Czech Science Foundation Grants 20-09628Y (A.B.) and 20-11563Y (T.F), Czech Ministry of Education grant Inter-COST LTC20074 (A. B.) and the European Regional Development Fund grant CZ.02.1.01/0.0/0.0/15_003/0000441 (T.F.).