Visualizing the living interface : bridging biophysical membrane properties and cellular communication

Cells constantly react to external stimuli to carry out a variety of functions. Recognizing and integrating these signals as well as subsequently mediating functional changes, requires highly sophisticated inter- and intracellular signaling networks, with immune signaling serving as a prime example. Initial events of many of these signaling cascades occur at the cell surface, and interfaces are formed between cells enabling cellular communication. Thereby the plasma membrane does not only act as a signaling hub by harboring receptors and ligands, but also has great impact on the dynamics of this interaction. Especially the biophysical properties of the plasma membrane, such as membrane fluidity, which are defined by the structure of the lipids it comprises, have a high impact on the signaling events. This interdisciplinary thesis explored the interplay of membrane biophysical properties and cell communication at cell-cell interfaces in the context of immune signaling. More specifically, the thesis aimed to characterize the sensitivities of the spectral shift as well as the fluorescence lifetime of fluorescent environment-sensitive dyes to distinct lipid environments by accurately applying confocal spectral imaging or FLIM, respectively. Regarding cell-cell interface formation, this thesis aimed to facilitate the study of individual proteins or glycocalyx components at these interfaces, e.g. the immune synapse, in 3D by developing two reconstitution model systems. Another aim was to pinpoint the underlying mechanism of mucin barrier removal during apoptosis and its effect on T cell efferocytosis. Individual sensitivities of environment-sensitive dyes Pro12A, NR12A, NR12S and Flipper to different lipid environments and therefore biophysical properties were unraveled both in confocal spectral imaging and FLIM. The lifetime of the dyes exhibited an emission-wavelength dependency most likely caused by solvent relaxation. Orientation and location of the dye within the membrane and their effect on the dipolar relaxation process of the dyes were identified as the underlying mechanisms for the individual sensitivities. A practicable protocol for membrane fluidity assessment with confocal spectral imaging was developed. Additionally, advantages, limitations and common pitfalls in data acquisition and analysis using confocal spectral imaging and FLIM were identified. To examine cellular communication in the context of immune signaling, two cell- cell interface reconstitution model systems were established, which enabled the study of ligand-receptor pairs such as adhesion proteins, pMHC-TCR interactions or checkpoint proteins. The semi-artificial system, allowed for examination of initial events in T cell activation, such as ZAP70 recruitment, and distinguished peptides of different affinity presented by the same MHC class I. Further, the segregation of heavily glycosylated proteins such as CD45, CD43 and MUC1 from the interfaces was observed. In apoptotic T cells however, most of these proteins were shed promoting the uptake by phagocytes. In apoptosis the flopping of PS to the exoplasmic leaflet activates the metalloprotease ADAM10 by introducing a conformational change, which then cleaves off the mucin ectodomains. Thus, removal or segregation of the mucin barrier alleviates a biophysical constraint in apoptotic cell clearance or cell-cell contact formation, respectively. Taken together, this thesis bridged the fields of membrane biophysics and molecular immunology. Environment-sensitive dyes are already widely used, however additional knowledge of their spectral shift and lifetime sensitivities as well as of potential technical pitfalls in confocal spectral imaging or FLIM allows researchers to select the best-suited dye for their individual research question. Further, this knowledge enables a more precise interpretation of the data, advancing our understanding of the influence of membrane biophysical properties in various contexts. Immune cell interfaces, such as the T cell immune synapse are of great interest as they are a possible point of therapeutic intervention for various diseases. The distribution of individual lipid, protein and glycocalyx components at the immune synapse can be examined with the established free-standing cell-cell interface reconstitution model systems. In fact, these model systems can be used to reconstitute virtually any cell-cell interface, highlighting their vast applicability. Further, the identification of glycocalyx regulation in apoptotic T cell clearance, highlights the influence of biophysical mechanisms such as size segregation on biological processes. List of scientific papers I. Ragaller F, Andronico L, Sykora J, Kulig W, Rog T, Urem YB, Abhinav, Danylchuk DI, Hof M, Klymchenko A, Amaro M, Vattulainen I, Sezgin E. "Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties". Open Biology. 2022 Sep;12(9):220175. https://doi.org/10.1098/rsob.220175 II. Ragaller F, Sjule E, Urem YB, Schlegel J, El R, Urbancic D, Urbancic I, Blom H, Sezgin E. "Quantifying Fluorescence Lifetime Responsiveness of Environment-Sensitive Probes for Membrane Fluidity Measurements". The Journal of Physical Chemistry B. 2024 Mar 7;128(9):2154-2167. https://doi.org/10.1021/acs.jpcb.3c07006 III. Carravilla P*, Andronico L*, Schlegel J*, Urem YB, Sjule E, Ragaller F, Weber F, Gurdap CO, Ascioglu Y, Sych T, Lorent J, Sezgin E. "Measuring plasma membrane fluidity using confocal microscopy". Nature Protocols. 2025 Jul;20(7):1976-2004. https://doi.org/10.1038/s41596-024-01122-8 IV. Ragaller F, Schneider AM, Sjule E, Sun R, Han X, Jenkins E, Dustin ML, Achour A, Sezgin E. "Studying macromolecular composition in cell- cell interfaces using 3D membrane reconstitution systems". bioRxiv 2025.11.01.686034. [Manuscript Preprint] https://doi.org/10.1101/2025.11.01.686034 V. Drexhage LZ*, Zhang S*, Dupont M, Ragaller F, Sjule E, Cabezas- Caballero J, Deimel LP, Robertson H, Russell RA, Dushek O, Sezgin E, Karaji N, Sattentau QJ. "Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis". Nature Communications. 2024 Jan 15;15(1):541. https://doi.org/10.1038/s41467-023-44619-8 * Authors contributed equally