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Development of fluorescence nanospectroscopy to elucidate the roles of nanoscale molecular segregation in the activation of T-cells

Periodic Reporting for period 1 - FNS-4-NAMOSAT (Development of fluorescence nanospectroscopy to elucidate the roles of nanoscale molecular segregation in the activation of T-cells)

Reporting period: 2016-10-01 to 2018-09-30

Understanding of molecular mechanisms of cellular processes is required in order to efficiently prevent, diagnose, and treat diseases. However, many of such key molecular events, including the first steps of the activation of the immune response, remain elusive due to the lack of experimental methodologies that could track these fast events on the molecular level in living cells. Nowadays, super-resolution fluorescence microscopy techniques allow us to non-invasively locate molecular structures of interest with a great precision, whereas spectroscopic methods provide rich information about molecular properties of the studied environment.
The main aim of this project was therefore to couple the strengths of super-resolution STED microscopy and of fluorescence spectroscopy employing environment-sensitive probes, into fluorescence nanospectroscopy (FNS), to offer new insights into the molecular arrangements, such as nanoscale lipid heterogeneities, in the membranes of T-cells upon their activation. We found out that the key membrane protein, the T-cell receptor (TCR), changes its preference for its membrane environment once engaged in signalling clusters, which importantly complements our understanding of the functional interplay between proteins and lipids in the membranes of living cells.
To achieve the goal above, we identified and characterised environment-sensitive membrane probes that are compatible with super-resolution STED microscopy. We demonstrated that the increased spatial resolution by STED enhances sensitivity to small environmental changes, such as small domains in model membranes and local differences in membrane order in live cells (Sezgin et al., Biophys. J., 2017). To further maximise the sensitivity, we integrated a multi-channel detector into a STED microscope, allowing us to record the shape of the fluorescent spectrum in any position of the image. Applying the method to immunology, we interrogated the differences in lipid order in the membranes of live activating T cells. Finding markedly different membrane properties at the sites of the key proteins involved in T-cell triggering, we provided important new insights into the molecular organisation of the initiation of the adaptive immune response.
The developed methodology to observe and characterise the very local lipid environment of membrane proteins is widely applicable to numerous fields of molecular and cell biology, such as signalling, trafficking, motility etc., and thereby importantly complements the information about the structure and molecular dynamics provided by super-resolution STED imaging and STED-FCS, respectively (Urbančič et al., Nano Letters, 2018; Urbančič et al., Viruses, 2018).
The specific findings about membrane organisation in activating T-cells (Santos et al., Nat. Immunology, 2018), contribute to clarifying the long-standing mystery of the roles of lipid heterogeneities in the initiation of the adaptive immune response. This might therefore elucidate the origins of the immune disorders (e.g. autoimmune diseases, allergies, and immune evasion of cancer) and hence help towards the design of new therapies, bringing long-term benefits to the society.