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.).