Actin and alpha-actinin dynamics in the adhesion and motility of EPEC and EHEC on host cells.
Two pathogenic Escherichia coli, Enteropathogenic E. coli (EPEC) and Enterohemorrhagic E. coli (EHEC), adhere to the outside of host cells and induce cytoskeletal rearrangements leading to the formation of membrane-encased pedestals comprised of actin filaments and other associated proteins beneath the bacteria. The structure of the pedestals induced by the two pathogens appears similar, although those induced by EHEC are shorter in length. Fluorescence Recovery After Photobleaching (FRAP) was used to determine potential differences of actin polymerization in EPEC and EHEC induced pedestals in cultured PtK2 cells expressing either Green or Yellow Fluorescent Protein (GFP or YFP) fused to actin or alpha-actinin. When all the fluorescent actin in a pedestal on EPEC-infected cells was photobleached, fluorescence recovery first occurred directly beneath the bacterium in a band that grew wider at a rate of one micron/minute. Consistently observed in all EPEC-induced pedestals, whether they were stationary, lengthening, or translocating, the rate of actin polymerization that occurred at the pedestal tip was approximately 1 mum/min. Overall, a much slower rate of actin polymerization was measured in long EHEC-induced pedestals. In contrast to the dynamics of GFP-actin, recovery of GFP-alpha-actinin fluorescence was not polarized, with the actin cross-linking protein exchanging all the length of the EPEC/EHEC induced pedestals. Surprisingly, the depolymerization and retrograde flow of pedestal actin, as well as pedestal translocations, were inhibited reversibly by either 2,3-butanedione monoxime (BDM) or by a combination of sodium azide and 2-deoxy D-glucose, leading to an increase in the lengths of the pedestals. A simple physical model was developed to describe elongation and translocation of EPEC/EHEC pedestals in terms of actin polymerization and depolymerization dynamics.
Shaner
Nathan C
NC
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6058, USA.
Sanger
Joseph W
JW
Sanger
Jean M
JM
D051339:Actin Depolymerizing Factors; D000185:Actinin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000494:pharmacology; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D004926:Escherichia coli / Q000472:pathogenicity*; D019453:Escherichia coli O157 / Q000472:pathogenicity*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D038761:Photobleaching; D048328:Potoroidae; D019810:Sodium Azide / Q000494:pharmacology
D051339:Actin Depolymerizing Factors; D000199:Actins; D008840:Microfilament Proteins; C485184:enhanced green fluorescent protein; D000185:Actinin; D049452:Green Fluorescent Proteins; C004717:diacetylmonoxime; D019810:Sodium Azide; D003847:Deoxyglucose; D003931:Diacetyl