Proteins in cells are not homogeneously distributed, but often localized to specific compartments, which may or may not be enclosed by membranes.  
Here I will first discuss the dynamic formation of membraneless compartments in the non-equilibrium environment of living cells and how this formation can be regulated, e.g. by kinases [1].  Then I will show how single-molecule FRET (smFRET) and tracking of biomolecules can be used to study conformational dynamics and time-resolved cellular localization in living cells [2]. Results with the transcription initiation factor TAF2 show that the same protein can be in different compartments and thereby have different functions [3]. For the heat shock protein Hsp90 we will determine how proteins may be recruited to different compartments and have different conformational dynamics.  
Altogether, smFRET has the potential to change our view on compartment-specific protein dynamics and therefore signalling within living cells.
 
[1] C. Lan, J. Kim, S. Ulferts, F. Aprile-Garcia, A. Anandamurugan, R. Grosse, R. Sawarkar, A. Reinhardt and T. Hugel, Nat. Commun., 14:4831 2023 (https://doi.org/10.1038/s41467-023-40540-2)
[2] A. Anandamurugan, A. Eidloth, P. Wortmann, L. Schrangl, F. Aprile-Garcia, C. Lan, R. Sawarkar, G. J. Schütz, T. Hugel, bioRxiv 2023 (https://doi.org/10.1101/2023.09.15.557875)
[3] T. Bhuiyan, P. K. Mendoza Sanchez, N. Arecco, J.  Kim, S. Nizamuddin, A. Prunotto, M. Tekman, M. L. Biniossek, S. Koidl, T. Hugel, S. J. Arnold, bioRxiv 2024, (https://doi.org/10.1101/2024.02.05.578926)