Enteropathogenic E. coli acts through WASP and Arp2/3 complex to form actin pedestals

D Kalman, OD Weiner, DL Goosney, JW Sedat… - Nature cell …, 1999 - nature.com
D Kalman, OD Weiner, DL Goosney, JW Sedat, BB Finlay, A Abo, JM Bishop
Nature cell biology, 1999nature.com
* Department of Microbiology and Immunology, GW Hooper Foundation Laboratories,
University of California at San Francisco, San Francisco, California 94143, USAś
Department of Biochemistry, University of California at San Francisco, San Francisco,
California 94143, USA# Biotechnology Laboratory, and the Department of Microbiology and
Immunology, University of British Columbia, Vancouver, BC V6T 1Z3 Canada** Onyx
Pharmaceuticals, Richmond, California 94806, USA† e-mail: kalman@ cgl. ucsf. edu‡ …
* Department of Microbiology and Immunology, GW Hooper Foundation Laboratories, University of California at San Francisco, San Francisco, California 94143, USAś Department of Biochemistry, University of California at San Francisco, San Francisco, California 94143, USA# Biotechnology Laboratory, and the Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3 Canada** Onyx Pharmaceuticals, Richmond, California 94806, USA
† e-mail: kalman@ cgl. ucsf. edu‡ These authors contributed equally to this work xtracellular stimuli can induce localized actin rearrangements at the site of stimulation. To understand how this occurs, we have been studying enteropathogenic Escherichia coli (EPEC), a bacterial pathogen that induces formation of an actin-rich membrane pseudopod or pedestal beneath itself upon adherence to host intestinal epithelia1. Infection ultimately results in diarrhoea, which can cause death, especially among infants in developing countries1. Here we show that pedestal formation depends on localized recruitment and activation of two host-cell factors involved in actin polymerization: the heptameric Arp2/3 complex (Arp2/3c), which nucleates polymerization2, and members of the Wiskott–Aldrich syndrome (WAS) family of proteins (WASP and N-WASP) 3, which bind to and activate Arp2/3c (ref. 2). Arp2/3c recruitment depends on WASP, and WASP recruitment depends on its GTPase-binding domain (GBD), suggesting involvement proximally of a Rho family GTPase. This is, to our knowledge, the first demonstration of cellular mediators of EPEC pedestal formation and of localized recruitment of WASP and Arp2/3c as part of a signalling cascade initiated at the cell surface. Exposure of HeLa cells to wild-type EPEC resulted in the formation of an actin-rich pedestal underneath the bacterium (see Supplementary Information). To determine whether endogenous WAS family proteins localized to the pedestals, HeLa cells were exposed to EPEC and then stained with a polyclonal antiserum that recognized WASP and N-WASP. At low magnification, pedestals are seen as punctate actin staining (Fig. 1c), directly apposed to the bacterium (Fig. 1a). The endogenous WASP-like protein (Fig. 1b) was enriched in the pedestal relative to the cell body. We next asked whether exogenous WAS family proteins affected pedestal formation or localized to pedestals. Flag-tagged wild-type WASP (WASP-WT) was expressed in HeLa cells, and the cells were exposed to EPEC. The EPEC are shown in Fig. 1e and the WASP-WT, detected by staining with the Flag antibody, in Fig. 1f. WASP-WT had no effect on pedestal formation, as measured by actin staining (Fig. 1g). Like the endogenous WAS-like protein, WASP-WT concentrated in pedestals (Fig. 1e–h, arrowheads). Haemagglutinin (HA)-tagged N-WASP behaved identically to WASP-WT (data not shown). Localization of transfected WASPWT to pedestals was specific: green fluorescent protein (GFP) fluorescence was not enriched in pedestals relative to the cell body (data not shown).
We determined whether WASP was required for pedestal formation by expressing various domains of WASP or N-WASP. Deletion of the WASP carboxy terminus (∆ C) results in pronounced defects in the capacity of the protein to polymerize actin4. Expression of WASP-∆ C blocked pedestal formation in a dominant-negative fashion (Fig. 1i–l). Pedestals, as measured by actin staining (Fig. 1k), were not evident beneath attached bacteria (Fig. 1i arrowheads) in cells expressing WASP-∆ C (Fig. 1j). Blockade occurred even in cells where the construct was expressed at low levels (Fig …
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