Cell-cell communication and signal transduction rely on the assembly of receptor-ligand complexes at
the plasma membrane. The spatiotemporal receptor organization plays a pivotal role in evoking cellular responses. Nevertheless, the mechanism for cluster formation and how its localization within the plasma membrane influence cell responses is not fully understood. Thus, tools which allow to control receptor networks with high spatiotemporal control are required. We developed tools to control neuropeptide Y2 hormone receptor (Y2R) and T-cell receptor (TCR) organization at the plasma membrane. Clustering triggered either via a chelator nanotool or via an ultrasmall photo-tool showed in situ Y2R enrichment in the clustered regions. We revealed a ligand-independent response by detecting increase in cytosolic calcium, cell spreading, and migration; signaling events which were analogous to the ones induced by the natural ligand. In another approach, we compare ligand-induced versus ligand-independent TCR clustering based on the interaction of a SpyTagged TCR and SpyCatcher protein arrays. Ligand-induced clustering in the confined regions exhibit higher density and dynamics than the ligand-free approach, indicating that the size and architecture of the pMHC ligand influences TCR assembly. These approached can be adapted to diverse receptor families raising the possibility to explore different modalities of receptor activation. 

M. Florencia Sánchez¹, Marina S. Dietz², Sevi Faria¹, Stefan Frühschulz¹, Lars Werkmann¹, Christian
Winter¹, Tina Karimian³, Sylvia Els-Heindl⁴, Annette G. Beck-Sickinger⁴, Ulrike Müller³, Julian
Weghuber^3,5, Karl Gatterdam¹, Ralph Wieneke¹, Birgit Plochberger^6,7, Mike Heilemann², Peter
Lanzerstorfer³, Robert Tampé¹

¹Institute of Biochemistry, Goethe University Frankfurt, Germany; ²Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Germany; ³School of Engineering and Environmental Sciences, University of Applied Sciences Upper Austria, Wels, Austria; ⁴Faculty of Life Sciences, Leipzig University, Germany; ⁵FFoQSI - Austrian Competence Centre for Feed and Food Quality, Safety & Innovation, Tulln, Austria; ⁶University of Applied Sciences Upper Austria, Campus Linz, Austria, ⁷Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria