Martinez-Arias Lab
Department of Genetics, University of Cambridge
Cassie Yu Bian
PhD student
yb228@cam.ac.ukDepartment of Genetics,
Downing Street, Cambridge
CB2 3EH,England
Telephone: +44 1223 766595
Fax: +44 1223 333992
The Notch protein contains an Extra-Cellular Domain (NECD), which contains a tandem array of up to 36 Epidermal Growth Factor (EGF)-like repeats that are involved in ligand interaction. Three juxtamembrane repeats known as Lin-12-Notch (LN) repeats modulate interactions between the NECD and the membrane-tethered Notch Intra-Cellular Domains (NICD), which includes seven ankyrin repeats flanked by nuclear localization signals, a PEST domain and a transactivation domain.
In the well characterised canonical Notch pathway, the signal is initiated by the binding of ligands, Delta or Serrate/Jagged, that triggers a series of cleavages that release NICD from the membrane into the nucleus, where it acts as a co-activator of the transcription factor CSL {for CBF in vertebrates, Suppressor of Hairless [Su(H)] in Drosophila and LAG-1 in C. elegans}to drive the transcription of target genes.
Notch signalling pathway have been reported to have common interactions with Wnt, and the laboratory of A. Martinez Arias has been pursuing this theme using Drosophila and more recently mouse embryonic stem cells, as model systems. In the course of this work, the laboratory of AMA has found that many elements of the Wingless/Wnt signalling pathway are linked to the Notch receptor biochemically and functionally. In particular it has been established that Notch targets an active form of Armadillo/ß-catenin, the effector of Wingless/Wnt signalling, for degradation and that in this manner it sets up a threshold for Wingless/Wnt signalling. Furthermore, it appears that this regulation is mediated by a specific aspect of the biology of the Notch receptor: its ligand independent traffic. The aim of my project is to link Notch and Armadillo/ β-catenin by characterizing Notch trafficking machinery.
Fig.1. Interaction of Notch trafficking and Wnt signalling pathway. Notch is endocytosed, at least, through two different routes, one ligand dependent, which leads to the generation of NICD and Su(H) dependent signalling (Fig6.1) and the other, ligand independent (Fig6.2a). Somewhere along this route their is an interaction with β-catenin/Armadillo which leads to its degradation or sequestration. Whether this is a branch of the ligand independent traffic of Notch is something that will be studied in this project. In the presence of Wnt signalling, the rate of Notch endocytosis is elevated and, probably through the action of Dsh, Armadillo/ß-catenin is liberated from Notch and, together with the activation of the canonical pathways, is allowed to interact with efficiently with TCF to promote Wnt dependent transcription.
The questions I aim to answer include:
- mapping ligand dependent and ligand independent traffic of Notch;
- what domains of Notch are involved in the traffic;
- linking Notch traffic and growth rate regulation;
- mapping the interaction between Notch and Armadillo inside the cell and characterizing the mechanism and consequences i.e. does Notch degrade, sequester or modify Arm? and
- if time allows, how Notch interacts with other signalling pathways
The techniques I am using include steady state antibody staining and pulse chase experiment.