Expanding the bioluminescent toolkit for in vivo imaging
Imaging tools have revolutionized our understanding of living systems by allowing researchers to visualize biological features in real time. Among the most popular imaging agents of this sort are the bioluminescent proteins (luciferases). These probes catalyze the oxidation of small molecule substrates (luciferins), releasing non-toxic, non-perturbing visible light in the process. Bioluminescence imaging (BLI) with luciferase-luciferin pairs is well suited for use in tissues and small animals. Since no excitation light is required, there is virtually no background signal. Indeed, BLI has been used to track numerous biological phenomena in vivo, including immune cell homing, tumor cell proliferation, and pathogen egress. Despite its remarkable versatility, bioluminescence lacks the spatial resolution necessary to monitor cell-cell interactions. Furthermore, BLI has been historically limited to monitoring one cell type or biological feature at a time. To address these issues, this dissertation involves engineering new luciferase tools and comprised two main goals: (1) engineering contact-dependent bioluminescent probes to report on cellular communication and (2) generating novel luciferase-luciferin pairs via directed enzyme evolution. Collectively, these tools will enable new studies of immune cell function, host-pathogen dynamics, and numerous other multi-cellular networks in vivo.