Change is the only constant Adapting high-content screening microscopy to study dynamic signaling mechanisms

Intracellular signaling pathways encode fundamental biological information through their precise temporal progression. However, traditional genetic screens rely on static endpoint readouts, leaving a gap in our understanding of dynamic signaling mechanisms. This thesis bridges this gap by developing and applying advanced optical screening platforms to resolve dynamic signaling phenotypes in mammalian cells, focusing on the cyclic adenosine monophosphate (cAMP) pathway as a model system. To capture these events, we utilize genetically encoded biosensors measured via Förster resonance energy transfer and fluorescence lifetime imaging microscopy (FRET-FLIM). The research first addresses challenges in imaging cell dynamics, identifying that widefield illumination causes photodegradation of β-adrenergic agonists, which could potentially lead to incorrect conclusions about cAMP signaling dynamics. By establishing confocal time-correlated single photon counting (TCSPC) as the superior recording paradigm, the study transitions from proof-of-concept arrayed siRNA screens to developing the first optical pooled screening (OPS) platform capable of resolving dynamic phenotypes in mammalian cells. Technical innovations, including a high-speed analysis pipeline (FAST-HIPPOS) and optimized tagging strategies, enable the simultaneous interrogation of thousands of genetic perturbations within a single imaging dataset. Finally, this robust technological framework is applied to uncover a novel mechano-signaling axis that goes via an adhesion GPCR (aGPCR) that couples force sensing to intracellular cAMP signaling and establishes itself as a bona fide mechanosensitive receptor. To conclude, the established dynamic OPS architecture can be adapted to interrogate any dynamic cellular process discernible by advanced light microscopy, transforming our capability to understand intricate cellular communication networks in human physiology.