Date of Completion
Molecular and Cell Biology
Cell Biology | Molecular Biology
Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne cause of bloody diarrhea and renal failure. During colonization of the intestine, EHEC injects the transmembrane receptor protein Tir and the cytoplasmic effector protein EspFU into host cells to reorganize the actin cytoskeleton into adhesion “pedestals.” EspFU has been shown to bind and activate the actin nucleation factor N-WASP to drive actin polymerization into pedestals. However, EspFU can still assemble pedestals in cells lacking N-WASP, suggesting that this effector protein is able to also trigger N-WASP-independent pathways of actin polymerization during infection. Cortactin is an atypical nucleation factor that localizes to pedestals, but its precise role in pedestal formation in the presence or absence of N-WASP has not been well defined. To test whether Cortactin functions in pedestal assembly, I used RNA interference to silence Cortactin expression in N-WASP wild type (WT) and N-WASP knockout (KO) mouse fibroblasts, infected these cells with an E. coli strain expressing myc-tagged EspFU and HA-tagged Tir, and examined them for actin pedestals. Consistent with previous observations, N-WASP KO cells contained slightly fewer and less-intensely stained actin pedestals than WT cells when each was treated with control siRNAs. Similarly, Cortactin depletion in WT cells resulted in a modest decrease in the number and intensity of actin pedestals. However, when Cortactin expression was silenced in KO cells, pedestal assembly was virtually abolished even though Tir and EspFU were still present. These results suggest that N-WASP and Cortactin are functionally redundant, and that Cortactin plays a previously-unrecognized potent role in N-WASP-independent pedestal assembly.
Grout, Sarah E., "Characterizing the Role of Cortactin in Actin Pedestal Assembly by Enterohemorrhagic Escherichia coli (EHEC)" (2013). Honors Scholar Theses. 308.