CELL BIOLOGY OF THE NEURON Polarity ... - Tavernarakis Lab

Cell Biology of the Neuron: Polarity, Plasticity and Regeneration, Crete 2011
In vivo Pull Down using a Synapsin-GFP-Neogenin
Transgenic Mouse Identifies Dock7 as a Novel
Neogenin Interacting Protein Involved in RGMa
Induced Axon Repulsion
Dianne van den Heuvel, Anita Hellemons, Jeroen Pasterkamp
Rudolf Magnus Institute of Neuroscience; UMC Utrecht; The Netherlands
Neogenin is a transmembrane receptor that regulates several key cellular
processes in the developing brain, including axon guidance, neuronal
differentiation, morphogenesis and apoptosis. In the process of neuronal network
formation binding of Repulsive Guidance Molecule A (RGMa) to Neogenin
elicits growth cone collapse and axon repulsion.
To further our understanding of the role and mechanism of Neogenin signalling in
the developing nervous system, we generated Synapsin-GFP-Neogenin transgenic
mice that express a GFP-Neogenin fusion construct under control of the neuronspecific
Synapsin I promoter. An in vivo anti-GFP pull down of GFP-Neogenin
from transgenic brain lysates followed by mass spectrometry identified Dock7 as
a novel interactor of Neogenin. Dock7 is a Rac GTPase-activating protein and an
important regulator of neuronal polarity with high expression levels in the
developing embryonic mouse brain.
In order to study the Neogenin-Dock7 interaction in more detail
immunohistochemistry was used to study protein colocalization both in mouse
embryonic brain slices and cultured primary cortex neurons.
n addition, co-immunoprecipitation experiments were performed using Neogenin
and Dock7 deletion mutants to identify the protein domains involved in the
interaction. On a functional level, we were able to show that knockdown of
Dock7 in mouse embryonic cortical neurons strongly reduces the axon repellent
effect of RGMa.
Presented by: van den Heuvel, Dianne
Poster No 114
Blue Session
196