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Abstract

Recording transient cellular events and biomolecular interactions on large scales is pivotal for understanding the mechanisms underlying diverse biological functions. This requires the development of innovative molecular tools that enable the massive parallel analysis of these phenomena in their natural context. To address this challenge, I developed a split version of HaloTag, a self-labeling protein that can be irreversibly labeled with a wide variety of fluorophore ligands. While both split-HaloTag fragments are inactive in isolation, their labeling activity is restored when brought into direct proximity by fusion to interacting proteins. Initially, I performed an in-depth characterization of HaloTag to gain insights into its biochemical properties, kinetics and substrate preferences. Building upon this knowledge, I engineered a split-HaloTag comprised of a large folded part and a small complementing peptide, and thoroughly characterized this split system to understand its properties and mechanism. By fusing the split-HaloTag parts to calcium-sensing proteins, I created a molecular recorder that becomes irreversibly labeled upon exposure to calcium, a universal second messenger in cellular signaling and indicator of neuronal activity. The recording window is defined by the presence of the fluorescent HaloTag ligand, and thus the successive usage of ligands with different colors enables recording of multiple epochs of calcium activity. The tool has facilitated the recording of brain-wide activity patterns in living flies and zebrafish larvae during visual stimulation in a follow-up study. I then engineered split-HaloTag to label interactions of synaptic adhesion proteins, in order to develop a highly selective method to stain neuronal connectivity. I used computational protein design methods to fine-tune the intrinsic affinity of the split-HaloTag fragments, and to increase the stability of the large fragment. The refined split-HaloTag successfully labels synaptic adhesion complexes at cell-cell interfaces, laying the foundation for a highly selective approach to large-scale connectivity mapping. Moreover, the usage of HaloTag ligands with different colors should permit the identification of newly formed synapses on a brain-wide scale. The excellent properties and versatility of the improved split-HaloTag system spurred the development of several other molecular recorders and labeling strategies in our group. Thus, the technology offers a promising pathway for exploring diverse biological activities and functions across various model systems.