Cdc42/N-WASP signaling links actin dynamics to pancreatic ß cell delamination and differentiation.

Delamination plays a pivotal role during normal development and cancer. Previous work has demonstrated that delamination and epithelial cell movement within the plane of an epithelium are associated with a change in cellular phenotype. However, how this positional change is linked to differentiation remains unknown. Using the developing mouse pancreas as a model system, we show that ß cell delamination and differentiation are two independent events, which are controlled by Cdc42/N-WASP signaling. Specifically, we show that expression of constitutively active Cdc42 in ß cells inhibits ß cell delamination and differentiation. These processes are normally associated with junctional actin and cell-cell junction disassembly and the expression of fate-determining transcription factors, such as Isl1 and MafA. Mechanistically, we demonstrate that genetic ablation of N-WASP in ß cells expressing constitutively active Cdc42 partially restores both delamination and ß cell differentiation. These findings elucidate how junctional actin dynamics via Cdc42/N-WASP signaling cell-autonomously control not only epithelial delamination but also cell differentiation during mammalian organogenesis.

N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination.

Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.

N-WASP is a novel regulator of hair-follicle cycling that controls antiproliferative TGF{beta} pathways.

N-WASP is a cytoplasmic molecule mediating Arp2/3 nucleated actin polymerization. Mice with a keratinocyte-specific deletion of the gene encoding N-WASP showed normal interfollicular epidermis, but delayed hair-follicle morphogenesis and abnormal hair-follicle cycling, associated with cyclic alopecia and prolonged catagen and telogen phases. The delayed anagen onset correlated with an increased expression of the cell-cycle inhibitor p21CIP, and increased activity of the TGFbeta pathway, a known inducer of p21CIP expression. Primary N-WASP-null keratinocytes showed reduced growth compared with control cells and enhanced expression of the gene encoding the cell-cycle inhibitor p15INK4B, a TGFbeta target gene. Inhibition of TGFbeta signaling blocked overexpression of p15INK4B and restored proliferation of N-WASP-deficient keratinocytes in vitro. However, induction of N-WASP gene deletion in vitro did not result in obvious changes in TGFbeta signaling or growth of keratinocytes, indicating that the in vivo environment is required for the phenotype development. These data identify the actin nucleation regulator N-WASP as a novel element of hair-cycle control that modulates the antiproliferative and pro-apoptotic TGFbeta pathway in keratinocytes in vivo and in vitro.

IRSp53 links the enterohemorrhagic E. coli effectors Tir and EspFU for actin pedestal formation.

Actin pedestal formation by pathogenic E. coli requires signaling by the bacterial intimin receptor Tir, which induces host cell actin polymerization mediated by N-WASP and the Arp2/3 complex. Whereas canonical enteropathogenic E. coli (EPEC) recruit these actin regulators through tyrosine kinase signaling cascades, enterohemorrhagic E. coli (EHEC) O157:H7 employ the bacterial effector EspF(U) (TccP), a potent N-WASP activator. Here, we show that IRSp53 family members, key regulators of membrane and actin dynamics, directly interact with both Tir and EspF(U). IRSp53 colocalizes with EspF(U) and N-WASP in actin pedestals. In addition, targeting of IRSp53 is independent of EspF(U) and N-WASP but requires Tir residues 454-463, previously shown to be essential for EspF(U)-dependent actin assembly. Genetic and functional loss of IRSp53 abrogates actin assembly mediated by EHEC. Collectively, these data indentify IRSp53 family proteins as the missing host cell factors linking bacterial Tir and EspF(U) in EHEC pedestal formation.

Role of the WASP family proteins for Mycobacterium marinum actin tail formation.

Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of inducing actin tail formation within the cytoplasm of macrophages, leading to actin-based motility and intercellular spread. Actin tail formation by M. marinum is markedly reduced in macrophages deficient in the Wiskott-Aldrich syndrome protein (WASP), which still contain the closely related and ubiquitously expressed protein N-WASP (neuronal WASP). In fibroblasts lacking both WASP and N-WASP, M. marinum is incapable of efficient actin polymerization and of intercellular spread. By reconstituting these cells, we find that M. marinum is able to use either WASP or N-WASP to induce actin polymerization. Inhibition or genetic deletion of tyrosine phosphorylation, Nck, WASP-interacting protein, and Cdc42 does not affect M. marinum actin tail formation, excluding the participation of these molecules as upstream activators of N-WASP in the initiation of actin-based motility. In contrast, deletion of the phosphatidylinositol 4,5-bisphosphate-binding basic motif in N-WASP eliminates M. marinum actin tail formation. Together, these data demonstrate that M. marinum subversion of host actin polymerization is most similar to distantly related Gram-negative organisms but that its mechanism for activating WASP family proteins is unique.

Pathogen-induced actin filament rearrangement in infectious diseases.

Host defence mechanisms involve the establishment and maintenance of numerous barriers to infectious microbes, including skin and mucosal surfaces, connective tissues, and a sophisticated immune system to detect and destroy invaders. Defeating these defence mechanisms and breaching the cell membrane barrier is the ultimate challenge for most pathogens. By invading the host and, moreover, by penetrating into individual host cells, pathogens gain access to a protective niche, not only to avoid immune clearance, but also to replicate and to disseminate from cell to cell within the infected host. Many pathogens are accomplishing these challenges by exploiting the actin cytoskeleton in a highly sophisticated manner as a result of having evolved common as well as unique strategies.

Enterohaemorrhagic and enteropathogenic Escherichia coli use different mechanisms for actin pedestal formation that converge on N-WASP.

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), two closely related diarrhoeagenic pathogens, induce actin rearrangements at the surface of infected host cells resulting in the formation of pseudopod-like structures termed pedestals beneath intimately attached bacteria. We have shown previously that N-WASP, a key integrator of signalling pathways that regulate actin polymerization via the Arp2/3 complex, is essential for pedestal formation induced by EPEC using N-WASP-defective cell lines. Here we show that actin pedestal formation initiated by EHEC also depends on N-WASP. Amino acid residues 226-274 of N-WASP are both necessary and sufficient to target N-WASP to sites of EHEC attachment. The recruitment mechanism thus differs from that used by EPEC, in which amino-terminal sequences of N-WASP mediate recruitment. For EPEC, recruitment of N-WASP downstream of Nck has been postulated to be mediated by WIP. However, we find a direct interaction of N-WASP with WIP to be dispensable for EPEC-induced pedestal formation and present data supporting an F-actin-dependent localization of WIP to actin pedestals induced by both EPEC and EHEC. In summary, our data show that EPEC and EHEC use different mechanisms to recruit N-WASP, which is essential for actin pedestal formation induced by both pathogens.

Phosphatidylinositol 4,5-biphosphate (PIP2)-induced vesicle movement depends on N-WASP and involves Nck, WIP, and Grb2.

Wiskott-Aldrich syndrome protein (WASP)/Scar family proteins promote actin polymerization by stimulating the actin-nucleating activity of the Arp2/3 complex. While Scar/WAVE proteins are thought to be involved in lamellipodia protrusion, the hematopoietic WASP has been implicated in various actin-based processes such as chemotaxis, podosome formation, and phagocytosis. Here we show that the ubiquitously expressed N-WASP is essential for actin assembly at the surface of endomembranes induced as a consequence of increased phosphatidylinositol 4,5-biphosphate (PIP2) levels. This process resulting in the motility of intracellular vesicles at the tips of actin comets involved the recruitment of the Src homology 3 (SH3)-SH2 adaptor proteins Nck and Grb2 as well as of WASP interacting protein (WIP). Reconstitution of vesicle movement in N-WASP-defective cells by expression of various N-WASP mutant proteins revealed three independent domains capable of interaction with the vesicle surface, of which both the WH1 and the polyproline domains contributed significantly to N-WASP recruitment and/or activation. In contrast, the direct interaction of N-WASP with the Rho-GTPase Cdc42 was not required for reconstitution of vesicle motility. Our data reveal a distinct cellular phenotype for N-WASP loss of function, which adds to accumulating evidence that the proposed link between actin and membrane dynamics may, at least partially, be reflected by the actin-based movement of vesicles through the cytoplasm.