Final Activity Report Summary - CORTACTINNMQ (Cortactin signalling and actin dynamics control in cell migration and bacterial invasion)
We have studied the role of cortactin function in pedestal formation by EPEC. As recently described Cortactin siRNA treated cells had reduced capability to support pedestal formation by EPEC. Moreover mutations of both the SH3 and NTA domain of cortactin interfere with pedestal formation. overexpression of an Erk-phosphorylated cortactin mimicking mutant exerted a neutral effect comparable to wt cortactin; whereas a Src-phosphorylated cortactin mimicking mutant blocked pedestal formation. However, a non-previously used erk non-phosphorylable mutant blocked pedestal formation similarly to the src non-phosphorylable mutant, which implicates, opposing the current view, that both phosphorylations exert a role on pedestal formation.
On other hand, cortactin and crk were shown to sinergise on actin polymerization during the invasion of Shigella, and that effect was dependent on the tyrosine phosphorylation of cortactin. We analysed if similar model could apply to EPEC infection. Using siRNAi and overexpression of a dominant negative mutant of crk, we demonstrated that Crk is not required for pedestal formation by EPEC. This result establishes a major signal transduction difference between Shigella and EPEC infection models.
We used purified recombinant cortactin and tir proteins to demonstrate a direct interaction of both and to characterise the effect of this interaction on actin polymerisation in vitro. Using nck and n-wasp K.O. cells we further analysed what type of complexes are de facto formed in vivo.
On other hand, cortactin and crk were shown to sinergise on actin polymerization during the invasion of Shigella, and that effect was dependent on the tyrosine phosphorylation of cortactin. We analysed if similar model could apply to EPEC infection. Using siRNAi and overexpression of a dominant negative mutant of crk, we demonstrated that Crk is not required for pedestal formation by EPEC. This result establishes a major signal transduction difference between Shigella and EPEC infection models.
We used purified recombinant cortactin and tir proteins to demonstrate a direct interaction of both and to characterise the effect of this interaction on actin polymerisation in vitro. Using nck and n-wasp K.O. cells we further analysed what type of complexes are de facto formed in vivo.