Our main goal is to understand chemotaxis: the complicated and fascinating process whereby cells move along chemical gradients. We also have a broad interest in the biology of the social amoeba Dictyostelium, as represented by our recent discovery of the mating-type locus and aspidocytes (environmentally resistant cells) as well as the previous identification of polyketide signals active in the social stage.
Chemotaxis must have arisen early eukaryotic evolution and now serves many purposes. It is vital in the immune system, for wound healing and in embryonic development and it allows Dictyostelium amoebae to chase bacterial prey or aggregate together. Based on evolutionary conservation, we use Dictyostelium as our main experimental tool to study chemotaxis because it offers great experimental advantages over all other systems.
The phosphoinositide lipids of the plasma membrane play a central role in chemotaxis. PIP3 forms gradients in the membrane, which can apparently trigger pseudopod formation.
PIP3 was once thought to act as a 'chemotactic compass' guiding cells up-gradient, but this idea is over-simplified as shown by the good chemotaxis of a mutant that cannot produce PIP3 gradients (this mutant lacks all five PI3kinases and the PTEN phosphatase that between them produce PIP3 gradients; Movie 3). We have recently found another mutant - one with greatly reduced PIP2 synthesis - which is much more severely chemotactically impaired than the PI3kinase/PTEN mutant, and one current project is to understand how PIP2 contributes to chemotactic signal transduction.
In this project we are producing a comprehensive description of the proteins that become phosphorylated in response to chemoattractant, their kinetics and sites of phosphorylation and, where possible, the protein kinases carrying out the phosphorylation. This project uses SILAC mass-spectroscopy and allows several thousand phospho-sites to be measured in one experiment. Around 100 proteins show large changes in phosphorylation. Many have been previously implicated in chemotaxis, but many are novel and are being investigated further.
We are also interested in a more mechanical aspect of how cells move.
The dominant view is that pseudopodia are extended by the force of actin polymerizing beneath the plasma membrane. A neglected alternative is that extension is driven by fluid pressure, resulting in the formation of blebs. Close observation shows that blebs frequently form at the leading edge of Dictyostelium cells, and can dominate under certain conditions, though in standard laboratory conditions blebs share the burden of extending the leading edge with F-actin driven projections. Bleb formation is chemotactic, and we are using both genetic screens and biochemistry to try and find out how blebs can be directed to form at a particular place by chemotactic gradients.
In earlier work we identified a chlorinated polyketide called DIF, and elucidated its biosynthetic and breakdown pathways and role in controlling cell differentiation during the multi-cellular stage of the life cycle. This work on polyketides continues, mainly in collaboration with other interested laboratories.
We recently discovered the mating type locus of Dictyostelium - the first one to be described from the Amoebozoa. Dictyostelium has three sexes, which are determined by the nature of the mating type locus. Just two genes are crucial and both encode small, soluble proteins without any known homologues in other mating systems. This discovery has opened many avenues, which are being pursued by Gareth Bloomfield, including the structural biology of the mating proteins and attempts to engineer the mating system into a useful tool for laboratory genetics.