Nck is just not involved in N WASP recruitment by EHEC. As an alternative, the EspFu Tccp effector activates N WASP, thereby mimicking Cdc42 signaling. Cantarelli et al. have proposed cortactin as the missing link connect ing TirEHEC and EspFu Tccp. They showed that EHEC initially induces tyrosine phosphorylation of cortactin after which induces its, similarly to the transient cortactin phosphorylation in the course of Helicobacter pylori infection. However, employing the two hybrid sys tem, they reported that tyrosine phosphorylated cortactin binds each TirEHEC and EspFu Tccp, and consistent with previously described binding assays applying recombinant purified proteins, only Erk phosphorylated cortactin binds N WASP. Current in vitro research using cells deficient in N WASP suggest that cortactin recruitment to EHEC pedestals occurs downstream of EspFu Tccp and N WASP.
It’s as a result essential to obtain additional insights into cortactin function in each systems. Important unresolved concerns contain no matter whether cortactin and TirEPEC interact directly, no matter if cortactin participates in the Tir Nck N WASP pathway, and selleck inhibitor how cortactin binding partners mod ulate its nucleating activity on pedestals. Hence, deepening our understanding from the involvement of cortactin on pedestals dynamics is relevant for many causes. Benefits Function of cortactin motifs in pedestal formation Reduction of cortactin expression by siRNA or over expression of its isolated SH3 domain, polyproline region or its helical region resulted in a drastic decrease in actin pedestal formation through infection with EPEC.
On the other hand the part of cortactins Arp2 three binding and acti vating region has not been addressed. Thus, we investigated its contribution to actin assembly on pedes tals using NVP-BEZ235 915019-65-7 EPEC to infect HeLa cells transiently transfected with GFP cortactin. Pedestals were visualized by immun ofluorescent staining of actin utilizing fluorescent phalloidin and bacteria with DAPI. As previously reported, no differences around the number of attached bacteria have been observed for the transfectants employed. The cortactin NTA domain carries a 20DDW22 motif that binds and activates the Arp2 3 complex. Mutation of this motif to 20DDA22, hereafter referred to as W22A, abol ished this activity. To figure out whether this motif is required for pedestal formation we transfected HeLa cells with GFP W22A. We utilised wild kind cortactin and GFP alone as controls.
As shown in Fig. 1, over expression of GFP FL cortactin permitted pedestal forma tion to levels equivalent to those in cells expressing GFP. Fig. 1C shows normalized percentages and stand ard deviations for GFP FL. Final results of 3 independent experiments were thought of statistically significant. Because the constructs bear a GFP tag we have been in a position to simultaneously assess the localization of various cortactin forms.