Dynamin is required for F-actin assembly and pedestal formation by enteropathogenic Escherichia coli (EPEC).
After attaching to human intestinal epithelial cells, enteropathogenic Escherichia coli (EPEC) induces the formation of an actin-rich pedestal-like structure. The signalling pathway leading to pedestal formation is initiated by the bacterial protein Tir, which is inserted into the host cell plasma membrane. The domain exposed on the cell surface binds to another bacterial protein, intimin, while one of the cytoplasmic domains binds the adaptor protein Nck. This leads to recruitment of other cytoskeletal proteins including neural Wiskott-Aldrich syndrome protein (N-WASP) and Arp2/3, resulting in focused actin polymerization at the site of bacterial attachment. In this study we investigated the role of the large GTPase dynamin 2 (Dyn2) in pedestal formation. We found that in HeLa cells, both endogenous and overexpressed Dyn2 were recruited to sites of EPEC attachment. Recruitment of endogenous Dyn2 required the presence of Tir, Nck and N-WASP but was independent of cortactin and Arp2/3. Knock-down of Dyn2 expression by RNA interference reduced actin polymerization and pedestal formation. Overexpression of dominant-negative mutants of Dyn2 also reduced pedestal formation and prevented recruitment of N-WASP, Arp3 and cortactin, but not Nck. Together, our results indicate that Dyn2 is an integral component of the signalling cascade leading to actin polymerization in EPEC pedestals.
Unsworth
Kate E
KE
Centre for Molecular Microbiology and Infection, Imperial College London, UK.
Mazurkiewicz
Piotr
P
Senf
Freya
F
Zettl
Markus
M
McNiven
Mark
M
Way
Michael
M
Holden
David W
DW
D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D034281:Dynamins / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D029968:Escherichia coli Proteins; D034281:Dynamins