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Production and characterisation of a tagged Notch library for use in live imaging of Notch receptor trafficking.
[Thesis]. Manchester, UK: The University of Manchester; 2019.
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Abstract
Notch signalling is a highly conserved signalling pathway; vital for both embryonic development and adult tissue homeostasis. Aberrant Notch signalling can result in both developmental abnormalities and disease pathogenesis. Such a core signalling pathway is highly regulated. This regulation influences which trafficking route the plasma membrane presented receptor subsequently takes. A key focus of the Baron lab is to better characterise the endocytic routes of Notch activation in Drosophila melanogaster, with an aim to visualise Notch signalling in real-time in living systems. This would greatly benefit the Notch field by shedding light on both the temporal regulation and localisation of the Notch receptor in response to different stimuli. To this end, the main aim of the project presented here is to develop and characterise the tools required to allow visualisation of both the extracellular and intracellular domains of the same Notch molecule, in real time. The Notch receptor is comprised of two main domains; the extracellular domain which functions as a receptor, and the intracellular domain which functions as a transcription factor. To date, the published fluorescently tagged Notch constructs are limited to labelling only one of these two domains. As the cleavage of the extracellular domain from the intracellular domain is a key step in Notch activation, the ability to observe both domains and how they traffic with respect to each other has the potential of providing better understanding of the fundamental basics of Notch signalling. The existing fluorescent Notch tools predominantly tag the transcriptionally active intracellular domain of Notch, with very few examples of tools which fluorescently tag the extracellular domain. This limitation is addressed here by the production and characterisation of the NBFP-GFP construct, which tags both the extracellular and intracellular domains of the same Notch molecule. NBFP-GFP behaves like wild-type Notch in terms of production of full-length protein, localisation to the membrane and transcriptional activity. The use of this construct is applied to live microscopy, and the relative localisation of both extracellular and intracellular domains to distinct endosomal domains is assessed in vitro. To better understand the roles of individual EGF-like repeats of the extensive extracellular EGF-domain of Notch, two cysteine mutant receptors (Nnd3 and NAxM1) are characterised in vitro. Finally, an example of how dual-tagged Notch tools can be applied to biological questions is provided. Inserting the notchoid 3 (nd3) mutation into NBFP-GFP showed that this mutation results in the apparent loss of the extracellular domain. This provides insight into how the nd3 mutation affects normal Notch trafficking as well as better understanding the role of the individual EGF-like repeats of the extracellular domain of Notch.