Enteropathogenic E-coli acts through WASP and Arp2/3 complex to form actin pedestals
Enteropathogenic E-coli acts through WASP and Arp2/3 complex to form actin pedestals
复制标题
DOI：
10.1038/14087
复制
发表时间：
1999-10-01
影响因子：
21.3
通讯作者：
Bishop, JW
中科院分区：
文献类型：
--
作者：
Kalman, D;Weiner, OD;Bishop, JW
*加州大学旧金山分校微生物与免疫学，GW Hooper基金会实验室，旧金山，加利福尼亚94143，美国；加州大学旧金山分校生物化学系，旧金山，加利福尼亚94143，美国#生物技术实验室，和微生物与免疫学，不列颠哥伦比亚大学，温哥华，BC V6T 1Z3加拿大** Onyx制药公司，里士满，加利福尼亚94806，美国；电子邮件：kalman@ cgl。加州大学旧金山分校。这些作者对这项工作的贡献相同，细胞外刺激可在刺激部位诱导局部肌动蛋白重排。为了理解这是如何发生的，我们一直在研究肠致病性大肠杆菌（EPEC），这是一种细菌病原体，在附着在宿主肠上皮上时，诱导自身下方形成富含肌动蛋白的膜伪足或基脚。感染最终导致腹泻，可导致死亡，特别是在发展中国家的婴儿中。在这里，我们发现基座的形成依赖于参与肌动蛋白聚合的两种宿主细胞因子的局部募集和激活：七聚体Arp2/3复合物（Arp2/3c），它形成聚合2，以及Wiskott-Aldrich综合征（WAS）蛋白家族成员（WASP和N-WASP） 3，它们结合并激活Arp2/3c（参考文献2）。Arp2/3c的募集依赖于WASP，而WASP的募集依赖于其GTPase结合域（GBD），提示其参与了Rho家族GTPase的近端。据我们所知，这是第一次证明EPEC基座形成的细胞介质，以及作为细胞表面启动的信号级联的一部分，WASP和Arp2/3c的局部募集。将HeLa细胞暴露于野生型EPEC导致在细菌下方形成一个富含肌动蛋白的基座（见补充信息）。为了确定内源性WAS家族蛋白是否定位于基底，将HeLa细胞暴露于EPEC，然后用识别WASP和N-WASP的多克隆抗血清染色。在低倍镜下，基座被视为点状肌动蛋白染色（图1c），直接与细菌相对（图1a）。内源性wasp样蛋白（图1b）相对于细胞体在基底富集。我们接下来询问外源WAS家族蛋白是否影响基座形成或定位于基座。在HeLa细胞中表达标记野生型WASP (WASP- wt)，并将细胞暴露于EPEC。EPEC见图1e，用Flag抗体染色检测的WASP-WT见图1f。通过肌动蛋白染色检测，WASP-WT对基座形成没有影响（图1g）。与内源性的was样蛋白一样，WASP-WT也集中在基座上（图1e-h，箭头）。血凝素（HA）标记的N-WASP表现与WASP-WT相同（数据未显示）。转染的WASPWT在基座上的定位是特异性的：相对于细胞体，基座上的绿色荧光蛋白（GFP）荧光并不富集（数据未显示）。我们通过表达WASP或N-WASP的不同结构域来确定WASP是否需要基座形成。WASP羧基端（∆C）的缺失导致该蛋白聚合actin - 4的能力出现明显缺陷。WASP-∆C的表达以显性-阴性的方式阻断了基底形成（图1 - 1）。通过肌动蛋白染色（图1k）测量，在表达WASP-∆C（图1j）的细胞中，附着细菌（图1i箭头）下没有明显的基座。即使在低水平表达的细胞中也会发生阻断（图2）。
* Department of Microbiology and Immunology, GW Hooper Foundation Laboratories, University of California at San Francisco, San Francisco, California 94143, USA¶ 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 …