Molecular and functional characterization of repulsive guidance by Netrin complexes

University Of Chicago, Chicago IL

Investigators

Abstract

Project Abstract: Neural connectivity, the collection of synapses wiring neurons, is a major property of a nervous system, and a determinant of neural function. In humans, billions of neurons make trillions of synapses, and the proper function of this system depends on proper wiring. Incorrect wiring of neurons can lead to improper perception and various neurodevelopmental diseases. While it is generally accepted that the connectivity is determined by cell surface receptors that uniquely label neurons and mechanistically guide their wiring, how these surface receptors signal is mostly uncharacterized. As one of the four classes of major axon guidance cues, UNC- 6/Netrins guide the direction of growth for many groups of neurons as studied in mammals, the fruit fly and the nematode C. elegans. Netrins can provide both attractive and repulsive cues to growing axons, which is dictated by the neuronal receptor: UNC-40/DCC for attractive responses, and UNC-5 for repulsive responses. The repulsive UNC-5 receptor family do not resemble in its domain composition and structure any of the other classes of structurally characterized guidance receptors, including the attractive UNC-40/DCC. Therefore, it is not clear how UNC-6/Netrin complexes with UNC-5 leads to repulsive signals, as opposed to the attractive UNC-6–UNC-40 complexes, despite three decades of genetic studies showing drastic phenotypes of misdirected growth of neurons in unc-5 mutants in several model organisms. The need for a structural understanding of UNC-6–UNC-5 complexes are further exacerbated by the fact that expression of unliganded UNC-5 can induce apoptosis, while UNC-6–UNC-5 complexes lead to over-proliferation of cells and are associated with cancer. To address this mechanistic gap in our understanding, we will develop biochemical strategies to constitute UNC-6–UNC-5 complexes and engineer these complexes through structure-guided rational strategies and directed evolution using yeast surface display. We propose to use the molecular tools and reagents we will develop for biophysical and structural characterization of UNC-5–UNC-6 and UNC-5– UNC-6–UNC-40 complexes, and to systematically characterize UNC-5 function and signaling using genetics in C. elegans and an in vitro cell death model in mammalian cell culture.