Development of Bioluminescent Biosensors Monitoring Activity of the Merlin Tumour Suppressor

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Pipchuk, Alex
Biosensor , Complementation Assay , Hippo Pathway , LATS , Luciferase , Merlin , NanoBiT , NanoLuc
All solid tumours share the ability to circumvent or overcome contact inhibition of proliferation (CIP), the process by which cell-cell contacts engage signalling to stop proliferation. Merlin, an ERM-like protein, plays a crucial role in CIP and is inactivated in a wide variety of cancers, suggesting that it is a tumour suppressor of general importance in cancer biology. Although the role of Merlin in cancer has been investigated extensively, this protein lacks intrinsic catalytic activity and has been notoriously difficult to study. To address this issue, we leverage novel luciferase technologies to develop and validate 2 split-luciferase biosensor systems that enable accurate quantification of Merlin activity in real time. First, we design an intramolecular biosensor aimed at studying Merlin’s changes in conformation state. In addition, we design a bimolecular split-luciferase system to investigate Merlin’s regulation of the Hippo pathway, a key downstream effector with well-established roles in cancer biology. Ultimately, these biosensors enable improved study of Merlin’s molecular functions, serving as powerful tools to investigate Merlin’s contributions to CIP and tumorigenesis.
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