NOVEL IRSP53 (INSULIN RECEPTOR SUBSTRATE PROTEIN OF 53 KDA) FUNCTIONS IN ACTIN CAPPING, FILOPODIA FORMATION AND POLARITY ESTABLISHMENT

IRSp53 (Insulin Receptor Substrate protein of 53 kDa) is a multidomain scaffolding protein that possesses an I-BAR domain (responsible for protein dimerization and membrane deformation), a partial CRIB motif (for the interaction with active CDC42), a SH3 domain (through it binds actin-related proteins) and additional protein interaction modules at the C-terminus. We have studied a previously unknown capping role of IRSp53 and the functional consequences of its interaction with VASP. In particular, we showed that following CDC42 binding and activation, IRSp53 is able to recruit and cluster VASP both in vitro and on the plasma membrane, thus fostering VASP processive elongation activity. The elongation of actin filaments by VASP, together with the membrane-deforming ability of IRSp53, combine to promote filopodia initiation downstream of CDC42. Thus, IRSp53 is essential for coordinating these activities in vitro and in cells in culture. In the second part of this thesis, an IRSp53 role in the polarity program was investigated. We showed that IRSp53 is apically localized in 3-D cyst models of epithelial cells, and IRSp53 depletion leads to a multilumen phenotype in MDCK and Caco2 spheroids. Moreover, using MDCK cysts as a model system, we noted that IRSp53 is enriched early at the prospective lumen initiation site (called AMIS), even before the apical marker podocalyxin. Finally, IRSp53 removal affects podocalyxin localization in MDCK cysts at the early stages. We speculate that IRSp53 might be involved in the trafficking of the apical protein podocalyxin, we are currently investigating the molecular mechanism responsible for this putative role.