Biophysical Society Thematic Meeting | Singapore

Mechanobiology of Disease

Poster Abstracts

71-POS Board 71 Role of Formin Fhod1 in Epec Pedestal Formation

Mrinal D. Shah 1 , G V. Shivashankar 1,4 , Alexander D. Bershadsky 1,3 , Linda J. Kenney 1,2 . 1 Mechanobiology Institute, Singapore, Singapore, 2 University of Illinois, Chicago, IL, USA, 3 Weizmann Institute of Science, Rehovot, Israel, 4 National University of Singapore, Singapore, Singapore. Enteropathogenic E. coli are extracellular pathogens that cause dense puncta of polymerized actin at the site of bacterial attachment referred to as ‘actin pedestals’. The polymerization of actin in the pedestal is thought to be solely regulated via the Nck-WASp-Arp2/3 pathway. Recent in vivo studies in mice have shown that Nck-deficient mice can still form actin pedestals, suggesting a redundancy in actin polymerization pathways and the possible involvement of multiple actin nucleators. It was therefore of interest to determine whether additional pathways involving the formins might play a role in the formation of actin pedestals in EPEC. We discovered that pedestal surface area was drastically reduced upon treatment with a small molecule formin inhibitor, SMIFH2, indicating a role for formins in the pedestal formation process. When we examined the localization of several classes of formins, we discovered a dense localization of formins FHOD1 and mDia1 at the base of the pedestal. In agreement with our result with formin inhibitors, we also observed that FHOD1 knockdown resulted in a similar size reduction of pedestals and this effect was restored by complementing FHOD1 on a plasmid. We also see interesting differences in the dynamics of Arp2/3 and FHOD1 in the pedestals, such that very large pedestals show a near absence of Arp3 intensity but very high FHOD1 intensity. This suggests a temporal segregation in the activity of Arp 2/3 and formins. Our findings suggest that EPEC uses multiple pathways in the process of pedestal formation and they probably act at different stages of pedestal the formation. We are now trying to understand the bacterial effectors that could be involved in this differential regulation and also its importance in the pathogenesis.

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