A yeast two-hybrid screen searched for novel interactions using a C-terminal fragment of Miranda as bait. This region (Mira 4) has been implicated in mutant studies as important for Miranda degradation in the GMC. The first screens yielded suggestive results but it was not possible to reproduce the interactions, probably due to a combination of low bait expression and high system background. Modifications to the system have produced a more robust screening protocol. <i>In vivo, </i>Mira4 confers instability on GFP, with expression levels of GFP-Mira4 much lower in the GMC than the neuroblast. Mira4 contains a KEN motif, a sequence recognised by APC/C<sup>Cdh1</sup>, and its mutation partially stabilises the construct. Full length GFP-tagged Miranda competes with endogenous Miranda, slowing both its degradation and also, interestingly, Pros release. Mutation of the KEN-box both relieves the competition, and increases the half-life of the GFP-fusion. Immuno-staining of wild-type embryos with a new Miranda antibody revealed some GMCs which retained cortical Miranda while Pros was entirely nuclear, showing Pros can be released without Mira degradation. Instead a model is proposed in which one signal is upstream of both events but that they lie in different pathways. The contribution of the KEN-box to Mira instability suggests the involvement of the APC/C although other E3 ligases cannot yet be ruled out. Biochemical assays to establish the ubiquitylation state of Miranda – by double immunoprecipitation and by affinity-binding to isolate ubiquitylated proteins – have also been developed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:599224 |
| Date | January 2006 |
| Creators | French, Catherine Louise |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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