N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility

Snapper, Scott B.; Takeshima, Fuminao; Antón, Inés M.; Liu, Ching-Hui; Thomas, Sheila M.; Nguyen, Deanna D.; Dudley, Darryll D.; Fraser, Hunter B.; Purich, Daniel L.; López-Ilasaca, Marco; Klein, Christoph; Davidson, Laurie A.; Bronson, Roderick T.; Mulligan, Richard C.; Southwick, Fred; Geha, Raif S.; Goldberg, Marcia B.; Rosen, Fred S.; Hartwig, John H.; Alt, Frederick W.

doi:10.1038/ncb1001-897

Cited by 309 publications

(314 citation statements)

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“…1D). Gross examination of E8.0-E11.0 embryos revealed marked developmental delay with prominent neural tube and cardiac abnormalities similar to those observed for N-Waspnull embryos (33). As early as E8.5, Abi-null embryos exhibited developmental delay (smaller size, some unturned embryos).…”

Section: Resultssupporting

confidence: 63%

Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

Dubielecka

Ladwein

Xiong

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.cell motility | lamellipodium | Rac | Arp2/3-complex | Hssh3bp1

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Section: Resultssupporting

confidence: 63%

Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

Dubielecka

Ladwein

Xiong

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been shown that human platelet extracts supported Shigella actin-based motility as added N-WASP in vitro (Egile et al, 1999). Recent reports using N-WASP-deficient mouse embryonic fibroblasts (Lommel et al, 2001;Snapper et al, 2001) confirmed our previous work indicating that N-WASP is required for Shigella motility in mammalian cells (Suzuki et al, 1998). However, the role of the WASP family proteins in Shigella spreading in infected cells such as epithelial cells or macrophages is poorly understood.…”

supporting

confidence: 58%

Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading

et al. 2002

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SummaryShigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by forming an actin comet tail. The VirG (IcsA) protein expressed at one pole of the bacterium recruits neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP family, which in turn stimulates actin-related protein (Arp) 2/3 complex-mediated actin polymerization. As all the WASP family proteins induce actin polymerization by recruiting Arp2/3 complex, we investigated their involvement in Shigella motility. Here, we show that VirG binds to N-WASP but not to the other WASP family proteins. Using a series of chimeras obtained by swapping N-WASP and WASP domains, we demonstrated that the specificity of VirG to interact with N-WASP lies in the N-terminal region containing the pleckstrin homology (PH) domain and calmodulin-binding IQ motif of N-WASP. A conformational change in N-WASP was important for the VirG-N-WASP interaction, as elimination of the C-terminal acidic region, which is responsible for the intramolecular interaction with the central basic region of N-WASP, affected the specific binding to VirG. We observed that, in haematopoietic cells such as macrophages, polymorphonuclear leucocytes (PMNs) and platelets, WASP was predominantly expressed, whereas the expression of N-WASP was greatly suppressed. Indeed, unlike Listeria, Shigella was unable to move in macrophages at all, (V) domain, a domain (C) with homology to the actindepolymerizing protein cofilin and, finally, a C-terminal acidic (A) segment (Miki et al., 1996). The C-terminal VCA domain interacts with the actin-related protein (Arp) 2/3 complex after the stimulation of actin polymerization by the complex (Rohatgi et al., 1999). N-WASP exists in two conformations: an inactive form in which the VCA domain is masked, and an active form in which it is exposed to promote actin polymerization by interaction with the Arp2/3 complex. The activation of N-WASP requires Cdc42 or PtdIns(4,5)P2 bound to N-WASP (Miki et al., 1998b;Rohatgi et al., 1999;2000;Prehoda et al., 2000). It has been reported that N-WASP is required for generating the actin tail from motile S. flexneri (Suzuki et al., 1998;Egile et al., 1999). Arp2/3 complex is also required for actin-based motility of Shigella, in which N-WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro (Egile et al., 1999).In mammalian cells, five WASP family members, N-WASP, WASP and WAVE1-3, have been identified and characterized to date (Suetsugu et al., 1999); however, none of them except N-WASP has been elucidated for involvement in the actin-based motility of Shigella in infected cells. It has been shown that human platelet extracts supported Shigella actin-based motility as added N-WASP in vitro (Egile et al., 1999). Recent reports using N-WASP-deficient mouse embryonic fibroblasts (Lommel et al., 2001;Snapper et al., 2001) confirmed our previous work indicating that N-WASP is required for Shigella motility in mammalian cells (Suzuki et al., 1998). However...

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“…In animal cells, the two major classes of Arp2͞3 activators are the WASP proteins and the Wave proteins. WASP proteins are dispensable for the formation of lamellipodia (2,3), whereas Wave proteins have emerged as a critical regulator of this structure. In fact, Wave proteins are localized at the tip of the protruding lamellipodium and delocalized whenever this structure retracts (4); genetic inactivation of the ubiquitously expressed Wave-2 gene causes defects in the formation of lamellipodia (5,6).…”

mentioning

confidence: 99%

Purification and architecture of the ubiquitous Wave complex

Gautreau

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

213

286

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The Wave proteins are major activators of the Arp2͞3 complex. The ubiquitous Wave-2 is required for actin polymerization at the leading edge of migrating cells. Here we purify Wave-2 from HeLa cells. Five proteins, Sra, Nap, Wave-2, Abi, and Hspc, are copurified, indicating that they form a tight complex. These proteins are only present in the complexed form, with the exception of Hspc, which displays a free pool. We reconstitute the Wave-2 complex by cotranslating in vitro the five subunits and use this system together with specific immunoprecipitations to study the molecular architecture of the complex. The complex is organized around a core of Nap and Abi. Sra is a peripheral subunit recruited on the Nap side, whereas the Wave and Hspc subunits are recruited on the Abi side of the core.

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N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility

Cited by 309 publications

References 50 publications

Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading

Purification and architecture of the ubiquitous Wave complex

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