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PubMed2003

Effects of ectopically expressed neuronal Wiskott-Aldrich syndrome protein domains on Rickettsia rickettsii actin-based motility.

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex have emerged as critical host proteins that regulate pathogen actin-based motility. Actin tail formation and motility in Listeria monocytogenes require the Arp2/3 complex but bypasses N-WASP signaling. Motility of Shigella flexneri and vaccinia virus requires both N-WASP and the Arp2/3 complex. Functional roles for these cytoskeletal regulatory proteins in actin-based motility of Rickettsia rickettsii have not been established. In this study, functional domains of N-WASP tagged with green fluorescent protein that have characterized effects on Shigella and vaccinia virus actin-based motility were ectopically expressed in HeLa cells infected with R. rickettsii to assess their effects on rickettsial motility. S. flexneri-infected cells were used as a control. Expressed N-WASP domains did not localize to R. rickettsii or their actin tails. Expression of N-WASP missing the VCA domain (for "verprolin homology, cofilin homology, and acidic domains"), which acts as a dominant-negative form of N-WASP, completely inhibited actin-based motility of S. flexneri while only moderately inhibiting motility of R. rickettsii. Similarly, expression of the VCA domain, which acts as a dominant-negative with respect to Arp2/3 complex function, severely inhibited actin-based motility of S. flexneri (no motility observed in the majority of expressing cells) but only moderately inhibited R. rickettsii motility. These results, taken together with the differential effects on motility observed upon expression of other N-WASP domains, suggest that actin-based motility of R. rickettsii is independent of N-WASP and the Arp2/3 complex.

Actin-Related Protein 2↗

PubMed1996

Studies of the expression of the Wiskott-Aldrich syndrome protein.

The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by thrombocytopenia, eczema, disorders in cell-mediated and humoral immunity, and a proclivity to lymphoproliferative disease. The gene responsible encodes a 53-kD proline-rich protein of unknown function (WASP). We produced a FLAG-WASP fusion protein that was used to immunize mice and produce mAbs against WASP. Using monoclonal anti-WASP in Western immunoblots, we have determined that WASP is present in the cytoplasmic but not nuclear fraction of normal human peripheral blood mononuclear cells, in normal human platelets, in T lymphocytes, non-T lymphocytes, and monocytes. The protein is produced in the B cell immunoblastic cell line DS-1, in normal EBV-transformed B cell lines, and in HEL92.1.7, but is barely detectable in MOLT-4 and not detectable in K562. WASP was present in two of four EBV-transformed cell lines from WAS patients. Splenic tissue immunostaining was performed in two patients, and the results correlated with the results of the Western blots. Sequence analysis of WASP cDNA from two patients who produce WASP show mutations causing amino acid substitutions. These studies establish a foundation for further studies aimed at understanding the function of WASP.

Amino Acid Sequence↗

PubMed2002

Gene therapy for Wiskott-Aldrich syndrome: rescue of T-cell signaling and amelioration of colitis upon transplantation of retrovirally transduced hematopoietic stem cells in mice.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that is caused by mutations in the recently identified WASP gene. WASP plays an important role in T-cell receptor-mediated signaling to the actin cytoskeleton. In these studies we assessed the feasibility of using retroviral gene transfer into WASP-deficient hematopoietic stem cells (HSCs) to rescue the T-cell signaling defect that is characteristic of WAS. Upon transplantation of WASP-deficient (WKO) HSCs that have been transduced with WASP-expressing retroviruses, mature B and T cells developed in normal numbers. Most importantly, the defect in antigen receptor-induced proliferation was significantly improved in T cells. Moreover, the susceptibility of colitis by WKO HSCs was prevented or ameliorated in recipient bone marrow chimeras by retrovirus-mediated expression of WASP. A partial reversal of the T-cell signaling defect could also be achieved following transplantation of WASP-deficient HSCs expressing the WASP-homologous protein N-WASP. Furthermore, we have documented a selective advantage of WT over WKO cells in lymphoid tissue using competitive repopulation experiments and Southern blot analysis. Our results provide proof of principle that the WAS-associated T-cell signaling defects can be improved upon transplantation of retrovirally transduced HSCs without overt toxicity and may encourage clinical gene therapy trials.

Animals↗

PubMed2005

Genotype-proteotype linkage in the Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a platelet/immunodeficiency disease arising from mutations of WAS protein (WASP), a hemopoietic cytoskeletal protein. Clinical symptoms vary widely from mild (X-linked thrombocytopenia) to life threatening. In this study, we examined the molecular effects of individual mutations by quantifying WASP in peripheral lymphocytes of 44 patients and identifying the molecular variant (collectively called proteotype). Nonpredicted proteotypes were found for 14 genotypes. These include WASP-negative lymphocytes found for five missense genotypes and WASP-positive lymphocytes for two nonsense, five frameshift, and two splice site genotypes. Missense mutations in the Ena/VASP homology 1 (EVH1) domain lead to decreased/absent WASP but normal mRNA levels, indicating that proteolysis causes the protein deficit. Because several of the EVH1 missense mutations alter WIP binding sites, the findings suggest that abrogation of WIP binding induces proteolysis. Whereas platelets of most patients were previously shown to lack WASP, WASP-positive platelets were found for two atypical patients, both of whom have mutations outside the EVH1 domain. WASP variants with alternative splicing and intact C-terminal domains were characterized for eight nonsense and frameshift genotypes. One of these, a nonsense genotype in a mild patient, supports expression of WASP lacking half of the proline-rich region. With one notable exception, genotype and proteotype were linked, indicating that a genotype-proteotype registry could be assembled to aid in predicting disease course and planning therapy for newly diagnosed infants. Knowledge of the molecular effect of mutations would aid also in identifying disease-modifying genes.

Adolescent↗

PubMed2002

Essential role of neural Wiskott-Aldrich syndrome protein in podosome formation and degradation of extracellular matrix in src-transformed fibroblasts.

Transformation of cells by the src oncogene causes dramatic changes in adhesive structures. In v-src-transformed 3Y1 rat fibroblasts (3Y1-src), there are actin-rich protrusive structures called podosomes by which attachment to the extracellular matrix is thought to occur. In this study, we found that neural Wiskott-Aldrich syndrome protein (N-WASP) colocalizes with filamentous actin (F-actin) in podosomes. Expression of dominant-negative mutants of N-WASP, Deltacof N-WASP and DeltaVPH N-WASP, both of which are incapable of activating the Arp2/3 complex, suppressed podosome formation, suggesting that N-WASP is essential in this process. Localization of N-WASP in podosomes appears to be attributable to interaction between N-WASP and the SH3 domain of cortactin. Indeed, microinjection of the cortactin SH3 domain suppressed podosome formation. We also observed that 3Y1-src cells cultured on fibronectin degrade the fibronectin primarily at the podosomes and that the inhibition of podosome formation by Deltacof N-WASP abolishes the fibronectin degradation. These results suggest the importance of N-WASP in podosome formation and extracellular matrix degradation, which are processes thought to underlie the invasive phenotype of 3Y1-src cells.

Actins↗

PubMed1993

Anaphylactoid shock following Hymenoptera sting as a presenting symptom of systemic mastocytosis.

Systemic mastocytosis is a rare and chronic disorder characterized by a pathologically increased number of mast cells in various tissues and overproduction of mast cell mediators. From a group of 15 patients (10 females, 5 males) with systemic mastocytosis five female patients presented with a history of an anaphylactoid shock reaction to wasp sting. Three of them had no demonstrable specific IgE against wasp or bee venom in serum, and a skin test that was only weakly positive for wasp venom. One patient had specific IgE against wasp venom and a clearly positive skin test to wasp venom. The other patient had specific IgE against both wasp and bee venom and a skin test that was only weakly positive to wasp venom. Two patients had to stop a hyposensitization procedure because of systemic side effects. The five patients did not differ from the other patients with systemic mastocytosis with regard to either clinical symptoms and signs or urinary excretion of histamine metabolites. From the latter group two female and three male patients said they had been stung by a wasp in the past. Thus, anaphylactoid shock after Hymenoptera sting can be a presenting symptom of systemic mastocytosis and may be caused by an IgE- as well as a non-IgE-mediated mechanism. In cases of anaphylactoid reaction to Hymenoptera sting, especially when there is no IgE demonstrable in serum or in cases of intolerance of hyposensitization, the diagnosis of systemic mastocytosis should be considered, also in the absence of the clinical hallmarks of urticaria pigmentosa.

Adult↗

PubMed2005

Immunoblot studies in allergic patients to hymenoptera venom before and during immunotherapy.

BACKGROUND: Hymenoptera venom immunotherapy (VIT) is immunologically effective in patients with systemic allergic reactions after hymenoptera stings. OBJECTIVE: To evaluate the effect of VIT on specific IgE and IgG4 immunoblotting bands in VIT-treated patients. MATERIAL AND METHODS: Specific IgE and IgG4 immunoblotting bands for hymenoptera venom were performed with ALABLOT in sera of 17 patients (8 allergic to honeybee venom, 8 to wasp and 1 to polistes venom) before and during successful VIT (1 and 3 years). Before immunotherapy, all patients had experienced moderate/severe systemic reactions to a hymenoptera sting, with positive skin tests and venom-specific IgE. During immunotherapy all patients suffered field stings, without any systemic reaction. RESULTS: Before VIT we detected several immunoglobulin-binding bands in different regions, with different individual patterns. After VIT, we observed in some patients (5/8 for honeybee venom, 6/8 for wasp and 1/1 for polistes) complete disappearance of some IgE-binding bands, mainly the 15 kDa region (honeybee) and 23 and 44 kDa regions (wasp and polistes). All patients showed decreased intensity of IgE-binding bands, most pronounced in regions 16, 44 and 52 kDa (honeybee); 44 and 35 kDa bands (wasp) and 23 kDa (polistes). Some patients showed de novo appearance of IgG4-binding bands (4/8 for honeybee and 8/8 for wasp venom), mainly in 52 kDa (honeybee) and in 23 and 44 kDa regions (wasp). All patients showed increased intensity of IgG4 bands that were already present before VIT, more pronounced in 52 and 44 kDa (honeybee) and in 44 and 35 kDa regions (wasp). CONCLUSIONS: During successful VIT there are changes in intensity and number of IgE and IgG4 binding bands, which could reflect the immunological improvement induced by VIT. These changes are more pronounced/frequent in wasp VIT, a fact that could explain the best results usually seen in these patients.

Adult↗

PubMed2004

Cuticular hydrocarbon dynamics in young adult Polistes dominulus (Hymenoptera: Vespidae) and the role of linear hydrocarbons in nestmate recognition systems.

In social insects, cuticular hydrocarbons (CHCs) play an important role in nestmate discrimination processes, but young individuals are usually not discriminated. We studied CHC changes in young workers of the social wasp Polistes dominulus. A quantitative estimation demonstrated that total quantities of CHCs increased after emergence, with branched alkanes increasing drastically when compared with other classes of hydrocarbons. The relative quantity of longer-chain compounds increased with respect to shorter ones; unsaturated compounds decreased. These changes might reduce the capacity of the cuticle to acquire compounds of environmental origin. We then tested whether individuals acquire hydrocarbons from the environment, and whether this capability equally characterises newly emerged and mature wasps. We exposed wasps of two age classes (adults younger or older than 24 h) to four linear hydrocarbons in turn, and observed how nestmates reacted to their re-introduction into the natal colony. Exposed young wasps elicited significantly more aggressive responses than control sisters; but treated wasps older than 24 h were generally accepted by nestmates. Chemical assays showed that exposed young wasps readily absorbed hydrocarbons; older ones did not incorporate hydrocarbons, suggesting that the chemical profiles of mature wasps are less prone to chemical shifts than those of newly emerged wasps.

Alkanes↗

PubMed1996

Presence of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in two Connecticut populations of Ixodes scapularis (Acari: Ixodidae).

Two Connecticut populations of the black-legged tick, Ixodes scapularis (Say), the vector of Lyme disease spirochetes, Borrelia bugrdorferi sensu stricto Johnson, Schmid, Hyde, Steigerwalt & Brenner, in the northeastern United States, are parasitized by the encyrtid wasp Ixodiphagus hookeri (Howard), formerly Hunterellus hookeri. The wasp was first detected in ticks from a forested site in Bridgeport in 1992. I. hookeri was reared from 18.6% of 148 host-seeking I. scapularis nymphs. In 1993 and 1994, this wasp was found to parasitize 26.0% of 192 engorged nymphs from Bridgeport and 21.8% of 101 nymphs from the Bluff Point Coastal Preserve in Groton. Each parasitized nymph produced an average of 6-8 wasps (range, 3-16) with a female to male sex ratio of 1.9-1. Both study sites are wooded, geographically isolated tracts (Bridgeport, 176 ha and Bluff Point, 326 ha) with high densities (51-72/km2) of white-tailed deer, Odocoileus virginianus (Zimmerman), and superabundant tick populations similar to that of Prudence Island, RI, where I. hookeri also has been reported. We found that I. hookeri emerged from 16.3% of 399 engorged nymphs and 13.7% of 1,081 engorged nymphs collected as unfed ticks from Prudence Island in 1990 and 1991, respectively. No wasps were obtained from nymphs collected in Stamford (n = 38) or Old Lyme, CT (n = 241). A high proportion (23.1% of 39) of engorged nymphs obtained from Bridgeport deer produced I. hookeri. However, only 2 nymphs (6.7%) recovered from white-footed mice, Peromyscus leucopus (Rafinesque), at Bridgeport were parasitized by the wasp. None of the engorged larvae recovered from deer or mice and fed as nymphs in the laboratory produced I. hookeri (n = 26 from deer and n = 384 from mice). The presence of this wasp in I. scapularis at these 2 insular-like sites on the Connecticut mainland supports the observation that high tick densities are required for the establishment and maintenance of I. hookeri and that the potential role of this wasp in the biological control of I. scapularis is limited.

Animals↗

PubMed1999

The pleckstrin homology domain of the Wiskott-Aldrich syndrome protein is involved in the organization of actin cytoskeleton.

In this study, we investigated the role of the pleckstrin homology (PH) domain of the Wiskott-Aldrich syndrome protein (WASP) in the regulation of actin cytoskeleton, which is defective in patients with Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Overexpression of the WASP in COS-7 cells cultured in the presence of fetal calf serum (FCS) resulted in large cluster formation of polymerized actin and WASP in the cytoplasm. In contrast, when the WASP transfected cells were cultured in the absence of FCS, activation with PMA or EGF was required to induce cluster formation. Overexpression of WASP with a missense mutation in the N-terminus of the PH domain failed to induce the large cluster formation in COS-7 cells even in the presence of FCS. We also found that phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is known to regulate the actin cytoskeleton, binds to the PH domain of WASP, and the binding was abolished by the introduction of a missense mutation into the N-terminus but not the C-terminus of the PH domain. Together with the observations that most of the missense mutations observed in patients with WAS and XLT are located within the PH domain, these results indicate that the PH domain of WASP plays important roles in the regulation of actin cytoskeleton and suggested that the binding of PIP(2) to the PH domain is necessary for WASP to function properly.

Actins↗

PubMed2006

The Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder with variable clinical phenotypes that correlate with the type of mutations in the WAS protein (WASP) gene. WASP, a key regulator of actin polymerization in hematopoietic cells, has 5 well-defined domains that are involved in signaling, cell locomotion, and immune synapse formation. WASP facilitates the nuclear translocation of nuclear factor kappaB and was shown to play an important role in lymphoid development and in the maturation and function of myeloid monocytic cells. Mutations of WASP are located throughout the gene and either inhibit or dysregulate normal WASP function. Analysis of a large patient population demonstrates a phenotype-genotype correlation: classic WAS occurs when WASP is absent, X-linked thrombocytopenia when mutated WASP is expressed, and X-linked neutropenia when missense mutations occur in the Cdc42-binding site. The progress made in dissecting the function of WASP has provided new diagnostic possibilities and has propelled our therapeutic strategies from conservative symptomatic treatment to curative hematopoietic stem cell transplantation and toward gene therapy.

Actins↗

PubMed2002

Flow cytometric determination of intracytoplasmic Wiskott-Aldrich syndrome protein in peripheral blood lymphocyte subpopulations.

We have produced a novel monoclonal antibody (mAb) directed against Wiskott-Aldrich syndrome protein (WASP) by immunizing mice with the recombinant protein. The mAb designated 5A5 is highly specific to WASP and suitable for Western blot analysis and immunoprecipitation. A flow cytometric assay using the 5A5 mAb identifies expression of intracytoplasmic WASP in lymphocytes from normal individuals. Double staining analysis with cell surface CD3, CD19, and CD56, and intracytoplasmic molecules revealed WASP expression in each subpopulation. With regard to WASP expression in patients with Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), peripheral blood mononuclear cells (PBMCs) from nine patients and Epstein-Barr virus-transformed B-lymphoblastoid cell lines from seven patients examined did not show WASP expression by flow cytometric analysis. These results were confirmed by Western blot analysis. We conclude that WASP expression in lymphocyte subpopulations from patients with WAS and XLT can be more precisely evaluated by flow cytometry as compared with Western blot analysis. This flow cytometry method is important as a supplement to Western blots, but even more important as an alternative and powerful assay that can contribute to research on WASP as well as diagnosis in a clinical setting.

Animals↗

PubMed1999

The Wiskott-Aldrich syndrome protein directs actin-based motility by stimulating actin nucleation with the Arp2/3 complex.

Actin polymerization at the cell cortex is thought to provide the driving force for aspects of cell-shape change and locomotion. To coordinate cellular movements, the initiation of actin polymerization is tightly regulated, both spatially and temporally. The Wiskott-Aldrich syndrome protein (WASP), encoded by the gene that is mutated in the immunodeficiency disorder Wiskott-Aldrich syndrome [1], has been implicated in the control of actin polymerization in cells [2] [3] [4] [5]. The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits [6] [7] [8], was recently shown to physically interact with WASP [9]. We sought to determine whether WASP is a cellular activator of the Arp2/3 complex and found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerized actin, formed actin tails and exhibited actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes were non-motile and exhibited only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization.

Actin-Related Protein 2↗

PubMed1999

Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes.

The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS null allele (WAS(-)(/)(-)). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44(-)CD25(+) to the CD44(-)CD25(-) stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-zeta, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH(2)-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS(-)(/)(-) cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS(-)(/)(-) lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS(-)(/)(-) neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.

Actins↗

PubMed2003

Rickettsial actin-based motility: behavior and involvement of cytoskeletal regulators.

Actin-based motility (ABM) is employed by spotted fever group (SFG) rickettsiae, such as Rickettsia rickettsii, to promote cell-to-cell spread. Time-lapse video microscopy revealed that ABM is not strictly confined to SFG rickettsiae as typhus group R. typhi moved at approximately the same rate as R. rickettsii (approximately 4 micro m/min), but in a highly erratic fashion. A number of common behaviors were observed between ABM of R. typhi and R. rickettsii, such as entrance into plasma membrane protrusions, formation of new actin tails only on the old surface of newly formed daughter cells, and quick (within 15 sec) reassembly of the actin tail to the opposite pole upon contact with cellular structures that impede forward movement. This last behavior suggests that the rickettsial protein(s) required for ABM is uniformly localized to both poles of the bacterium and possibly throughout the rickettsial surface. Functional roles in rickettsial ABM for neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex, critical regulators of ABM of other pathogens, have not been established. Domains of N-WASP that have characterized inhibitory effects on N-WASP or Arp2/3 complex function were expressed in HeLa cells infected with R. rickettsii. Shigella flexneri-infected cells were used as a control. When ectopically expressed, the VCA domain of N-WASP (VCA) acts as a dominant/negative with respect to Arp2/3 complex function and N-WASP missing VCA (DeltaVCA) acts as a dominant/negative form of N-WASP. Expression of VCA or DeltaVCA severely inhibited S. flexneri ABM (no Shigella motility observed in the majority of expressing cells) while only moderately inhibiting ABM of R. rickettsii (approximately 35% decrease in the rate of ABM). In addition, ectopically expressed full-length GFP-N-WASP was recruited by S. flexneri but not R. rickettsii, and Arp3 was detected by indirect immunofluorescence in S. flexneri actin tails but not within R. rickettsii actin tails. Collectively, these data suggest that rickettsial ABM is independent of N-WASP and Arp2/3 complex function.

Actins↗

PubMed2003

SLP-76 coordinates Nck-dependent Wiskott-Aldrich syndrome protein recruitment with Vav-1/Cdc42-dependent Wiskott-Aldrich syndrome protein activation at the T cell-APC contact site.

We have shown previously that Wiskott-Aldrich syndrome protein (WASP) activation at the site of T cell-APC interaction is a two-step process, with recruitment dependent on the proline-rich domain and activation dependent on binding of Cdc42-GTP to the GTPase binding domain. Here, we show that WASP recruitment occurs through binding to the C-terminal Src homology 3 domain of Nck. In contrast, WASP activation requires Vav-1. In Vav-1-deficient T cells, WASP recruitment proceeds normally, but localized activation of Cdc42 and WASP is disrupted. The recruitment and activation of WASP are coordinated by tyrosine-phosphorylated Src homology 2 domain-containing leukocyte protein of 76 kDa, which functions as a scaffold, bringing Nck and WASP into proximity with Vav-1 and Cdc42-GTP. Taken together, these findings reconstruct the signaling pathway leading from TCR ligation to localized WASP activation.

Actins↗

PubMed2002

Neural Wiskott-Aldrich syndrome protein is involved in hepatocyte growth factor-induced migration, invasion, and tubulogenesis of epithelial cells.

Neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP family, regulates reorganization of the actin cytoskeleton through activation of the Arp2/3 complex. To date, most studies of N-WASP have focused on intracellular and morphological phenomena, such as vesicle transport and filopodium formation. We investigated the importance of N-WASP in epithelial morphogenesis, using Madin-Darby canine kidney epithelial cells, which form branching tubules when cultured with hepatocyte growth factor (HGF) in collagen gel. We established MDCK cell lines that overexpress wild-type N-WASP (WT-NW) or a dominant-negative form of N-WASP (DN-NW). WT-NW and parental Madin-Darby canine kidney cells formed branching tubules in collagen gel in response to HGF. However, formation of branching tubules was suppressed in DN-NW cells. During tubulogenesis, endogenous N-WASP accumulated at cell extensions protruding from the walls of the cysts and at the tips of the extending tubules. Gross cell morphology, cell-cell adhesion, cell polarity, and scattering in response to HGF were unaffected in WT-NW and DN-NW cells. In contrast, directed cell migration and HGF-induced invasion were significantly repressed in DN-NW cells. These results indicate that N-WASP regulates HGF-induced cell migration and invasion, which are required for epithelial tubulogenesis.

Animals↗

PubMed2005

Parasitic suppression of feeding in the tobacco hornworm, Manduca sexta: parallels with feeding depression after an immune challenge.

The parasitic wasp, Cotesia congregata, suppresses feeding in its host Manduca sexta. Feeding suppression in the host coincides with the emergence of the wasps through the host's cuticle. During wasp emergence, host hemocyte number declined, suggesting that the host mounts a wound/immune response against the exiting parasitoids and/or resulting tissue damage. Eliciting a different type of immune response by injecting heat-killed Serratia marcescens also resulted in a decline in feeding and a reduction in hemocyte number. Both the emerging wasps and the bacteria induced an increase in hemolymph octopamine concentration and a decrease in foregut peristalsis in M. sexta. The emerging parasitoids produced the largest changes. The source of the additional octopamine appeared to be the host in both cases. S. marcescens was found to contain no detectable amounts of octopamine. The parasitoids had insufficient octopamine to account for the amount found in host hemolymph and they did not secrete octopamine in vitro. One cause for the high concentration of octopamine in parasitized M. sexta was that octopamine was removed from the hemolymph approximately 23 times more slowly after the wasps emerged than prior to wasp emergence. The striking similarity between the effects of parasitoids and bacteria on M. sexta feeding, hemocyte number, hemolymph octopamine concentration, and foregut peristalsis supports the possibility that the immune/wound reaction induced by the emerging wasps could play a role in the suppression of host feeding. These results also support the hypothesis that M. sexta exhibit an immune-activated anorexia.

Animals↗