Research objectives and content Little is known about actin nucleation in non-muscle cells, a process usually associated with membranes. It is often induced by signals from outside the cell and some phosphoinositides, especially PIP, PIP2 and PIP3 seem to be important. Moreover, many actin binding proteins can bind these lipids. Recently a simple in vitro assay was developed by the Griffiths group to study actin nucleation on purified phagosomal membranes. Actin nucleation in this system is dependent on PIP2 and on peripheral membrane proteins that are removed by 'high salt' treatment. This process can be reconstituted by adding 'high salt' extracts to salt-stripped phagosomes. Further, a correlation was found between the age period in which phagosomes can fuse (in a separate assay) and that in which they best nucleate actin. I propose to identify and characterize the protein(s) which nucleate(s) actin on phagosomal membranes and to try to correlate its (their) function with the fusion process. Training content (objective, benefit and expected impact) At the EMBL I can learn and apply the use of an elegant system that is highly versatile to study membrane-actin interactions as well as membrane fusion in a (for me) new field. Links with industry / industrial relevance (22) A deeper understanding of these processes is a fundamental cell biological problem that is important for all pharmaceutical companies.