The role of annexin 1 in mediating inward vesiculation within multivesicular endosomes. (360G-Wellcome-078304_Z_05_Z)

We have shown that EGF stimulation promotes multivesicular endosome/body (MVB) formation and inward vesiculation within MVB. MVB formation requires the ESCRT1 component, TSG101, whilst EGF stimulated inward vesiculation within MVB is totally dependent on tyrosine phosphorylation of annexin 1. EGF stimulation promotes the association of annexin 1 with MVB and annexin 1 accumulates with the EGFR on the internal vesicles of MVB. We propose to determine what regulates the enhanced association of annexin 1 with MVBs in EGF-stimulated cells, focusing upon the roles of the N-terminal domain of annexin 1, association of annexin 1 with the EGF receptor and interactions of annexin 1 with inositol phospholipids. We will then determine the molecular mechanisms underlying annnexin 1-mediated inward vesiculation. We will determine the role of the N-terminus of annexin 1 and whether the annexin 1 ligand, S100-A11, or proteolysis of annexin 1 are required. By performing qualitative and quantitative electron microscopy on annexin 1 knockout cells in which annexin 1 mutants have been expressed, and on wild type cells in which dominant negative annexin 1 mutants have been expressed, we will determine at which step(s) in the inward vesiculation process annexin 1 operates.