Biochemistry of novel neural protein complexes

http://www2.mrc-lmb.cam.ac.uk/groups/debono/

Our goal is to understand how nervous systems generate behaviour at molecular, cellular, and circuit levels. Using C. elegans forward genetics we have identified a series of highly conserved proteins that are expressed predominantly in the nervous system, and are associated with behavioural phenotypes, but whose functions are poorly understood in any animal.

The mutant phenotypes of these genes, assessed by behavioural assays, neural imaging, and cell biology, hint at what these proteins do, but biochemistry is required to establish their functions. The goal of this project is to use biochemical approaches to identify and study complexes formed by these proteins.

For example, two proteins form an ER complex required for biogenesis of GPCRs. A different ER protein is required for depolarization of some neurons, suggesting a role in the biogenesis or function of an ion channel. Several are polytopic proteins expressed at the surface of neurons. Others are soluble cytoplasmic proteins previously studied in the context of the mammalian immune responses, but uncharted in the nervous system.

C. elegans is well-known for its powerful genetics, the wiring diagram of its nervous system, and its tractability for in vivo microscopy. Less appreciated are its advantages for biochemistry. Proteins can be rapidly tagged in vivo for immunoprecipitation followed by mass spec analysis; gram quantities of worms can easily be grown in a few days; and efficient protocols to extract protein complexes are available. Biochemistry complements genetics – we expect to find that some neural proteins plug into complexes required for viability that are inaccessible to screens for behavioural phenotypes.

The project envisages an emphasis on biochemistry using C. elegans and tissue culture cells, but experiments will constantly refer back to in vivo analysis, e.g. using CRISPR to knockout interacting proteins and examining their mutant phenotypes using different assays.