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At least 199 records · Page 11Linked to original sources
PubMed1997

Developmental changes in teratocytes of the braconid wasp Cotesia congregata in larvae of the tobacco hornworm, Manduca sexta.

The endoparasitic wasp Cotesia congregata develops in the hemocoel of larval stages of the tobacco hornworm, Manduca sexta. Teratocytes were released from the serosal membrane during hatching of the first instar wasp larva at 2-3days after oviposition; about 160 cells were released per embryo. The cells increased in diameter from about 10 to >200&mgr;m prior to wasp emergence. Nascent microvilli, visible on the cell surface before hatching of the first instar larva, rapidly increased in length and number following release of the cells. Irrespective of when the wasps were due to emerge, or how many parasitoids were present in the host, dramatic cytological changes occurred in the cells during the last instar of the host's development. Many of these morphological and ultrastructural changes were symptomatic of the cytological features of degenerating or apoptotic cells, and large numbers of vesicles appeared interspersed amongst the microvilli. The nucleus developed extensive dentritic ramifications, and the chromatin condensed in large clumps on the inner nuclear membrane. At the final stages of the wasps' development, the nucleus occupied the bulk of the interior of the cell. The cytoplasm gradually grew dramatically more electronluscent and less granular, as did the nucleoplasm, which is also indicative of impending cell death. Following the parasites' emergence, many of the cells underwent extensive blebbing of the cell surface. Teratocytes within a host appeared heterogeneous with respect to their morphological appearance. Analysis of the proteins secreted by teratocytes in vitro following labelling with (35)S-methionine showed that many (>30) polypeptides were synthesized de novo and secreted by the cells; some proteins were clearly targeted for secretion. We presume that the cells likely secrete a large number of proteins in vivo as well as in vitro.

Journal Article↗
PubMed1999

Effect of food quality on the body temperature of wasps (Paravespula vulgaris).

Body surface temperature of individually marked wasps (Paravespula vulgaris, Vespidae, Hymenoptera) was measured by infrared thermography during repeated visits to a feeding bowl without injuring them or disturbing their behavior. Wasps were fed 0.5, 1 and 2 mol/l sucrose solution at two ambient temperatures.Thoracic temperature varied significantly in dependence on food quality (sucrose concentration of solution). At the higher ambient temperatures of 26.1-30.2 degrees C mean thoracic surface temperatures from different experiments were 35.3 and 38.0 degrees C when the wasps took a 0.5 mol/l sucrose solution, 37.0, 38.7 and 38.7 degrees C when they took a 1 mol/l solution, and 39.1 degrees C when they took a 2 mol/l sucrose solution. At the lower ambient temperatures of 17.6-21.0 degrees C thoracic temperatures were lower but the effect of different sucrose concentrations was similar: 34.7 degrees C with a 0.5 mol/l and 36.1 degrees C with a 1 mol/l sucrose solution. The concentration effect amounted to about 10-25% of the whole variability of thorax temperature. By contrast, the temperatures of the head and abdomen did not follow the changes in thorax temperature according to changes in sucrose concentration closely, which suggests that the pattern of haemolymph circulation may have changed after landing, during the wasps' stay at the feeder. At initial landing at the feeders thoracic temperatures where equal to (three of eight tests) or lower (five of eight tests) than at final departure.The correlation of thorax temperature with food quality probably reflects the wasps' level of excitement and motivation to collect the food, which allows them to balance energetic investment with profitability of foraging and the needs of flight muscle performance and motility.

Journal Article↗
PubMed2006

Biochemical properties and inhibitors of (N-)WASP.

The Wiskott-Aldrich syndrome protein (WASP) is an effector of the Rho GTPase Cdc42 and a key component of signaling pathways that regulate the actin cytoskeleton. WASP is regulated by a number of ligands, and the mechanisms by which these act are beginning to be understood through detailed biochemical analyses. Here we describe the protocols we use to study WASP proteins, including the methods we use to purify signaling components and the assays we use to quantitatively characterize the biochemical and biophysical properties of WASP, its activation by Cdc42, and its inhibition by the small molecule wiskostatin. These methods have broad use within the WASP-related cytoskeletal-signaling pathway but are also applicable to investigations of other intramolecular and intermolecular interactions.

Actin-Related Protein 2-3 Complex↗
PubMed1996

Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases.

BACKGROUND: Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins, and a number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signal-transduction pathways, and the purpose of this work was to identify SH3-binding proteins in haematopoietic cells. RESULTS: We performed affinity-chromatography experiments with a panel of GST-SH3 fusion proteins (composed of glutathione-S-transferase appended to various SH3 domains) to search for SH3-binding proteins in a human megakaryocytic cell line. Protein microsequencing identified one of the SH3-binding proteins as WASp, the protein that is defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WASp bound preferentially in vitro to SH3 domains from c-Src family kinases, and analysis of proteins expressed in insect cells using a baculovirus vector demonstrated a specific interaction between WASp and the Fyn protein-tyrosine kinase. Finally, in vivo experiments showed that WASp and Fyn physically associate in human haematopoietic cells. CONCLUSIONS: Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction, including reduced proliferation and tyrosine phosphorylation in response to stimulatory factors. Members of the c Src family of protein-tyrosine kinases, including Fyn, are involved in a range of signalling pathways - such as those regulating cytoskeletal structure - in both haematopoietic and non-haematopoietic cells. Our data suggest that binding of Fyn to WASp may be a critical event in such signalling pathways in haematopoietic cells.

Blotting, Western↗
PubMed2003

Negative regulation of yeast WASp by two SH3 domain-containing proteins.

BACKGROUND: WASp family proteins promote actin filament assembly by activating Arp2/3 complex and are regulated spatially and temporally to assemble specialized actin structures used in diverse cellular processes. Some WASp family members are autoinhibited until bound by activating ligands; however, regulation of the budding yeast WASp homolog (Las17/Bee1) has not yet been explored. RESULTS: We isolated full-length Las17 and characterized its biochemical activities on yeast Arp2/3 complex. Purified Las17 was not autoinhibited; in this respect, it is more similar to SCAR/WAVE than to WASp proteins. Las17 was a much stronger activator of Arp2/3 complex than its carboxyl-terminal (WA) fragment. In addition, actin polymerization stimulated by Las17-Arp2/3 was much less sensitive to the inhibitory effects of profilin compared to polymerization stimulated by WA-Arp2/3. Two SH3 domain-containing binding partners of Las17, Sla1 and Bbc1, were purified and were shown to cooperate in inhibiting Las17 activity. The two SLA1 SH3 domains required for this inhibitory activity in vitro were also required in vivo, in combination with BBC1, for cell viability and normal actin organization. CONCLUSIONS: Full-length Las17 is not autoinhibited and activates Arp2/3 complex more strongly than its WA domain alone, revealing an important role for the Las17 amino terminus in Arp2/3 complex activation. Two of the SH3 domain-containing ligands of Las17, Sla1 and Bbc1, cooperate to inhibit Las17 activity in vitro and are required for a shared function in actin organization in vivo. Our results show that, like SCAR/WAVE, WASp proteins can be controlled by negative regulation through the combined actions of multiple ligands.

Actin-Related Protein 2↗
PubMed2001

Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.

Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site.

Antigen-Presenting Cells↗
PubMedDate not supplied

Wiskott-Aldrich syndrome protein, WASP.

Wiskott-Aldrich Syndrome protein (WASP) is the product of the gene mutated in children with Wiskott-Aldrich Syndrome (WAS). It is a predominantly cytoplasmic protein, expressed only in haematopoietic cells. It binds in vivo to the adaptor proteins Nck and Grb2, to the cytoplasmic protein-tyrosine kinase Fyn and to the small Rho-like GTPase Cdc42, which is required for formation of filopodia in fibroblasts and macrophages. WASP also interacts, directly or indirectly, with the actin cytoskeleton. Together with studies of a closely related, ubiquitously expressed protein named N-WASP, these findings suggest that WASP is a component of signalling pathways that control reorganisation of the actin cytoskeleton in haematopoietic cells in response to external stimuli. In support of this idea, haematopoietic cells from WAS patients show defects in cytoskeletal organisation that compromise their ability to polarise and to migrate in response to physiological stimuli. These defects could account for many of the clinical features of WAS. WAS is now a candidate for gene therapy based on the delivery of a wild-type WASP gene to autologous haematopoietic stem cells. In addition, recent studies of cell defects in WAS patients suggest that it may prove possible, in time, to rescue WAS cells using more conventional drug therapies.

Humans↗
PubMed2001

Global disruption of the WASP autoinhibited structure on Cdc42 binding. Ligand displacement as a novel method for monitoring amide hydrogen exchange.

The Cdc42 GTPase, a member of the Rho subfamily of Ras proteins, can signal to the cytoskeleton through its effector, the Wiskott-Aldrich syndrome protein (WASP), activation of which results in localized polymerization of new actin filaments. NMR structures of WASP peptide models in the Cdc42-bound and free states suggest that GTPase binding weakens autoinhibitory contacts between the GTPase binding domain (GBD) and the C-terminal actin regulatory (VCA) region of the protein. In the study presented here, amide hydrogen exchange has been used with NMR spectroscopy to directly examine destabilization of the autoinhibited GBD-VCA conformation caused by GTPase binding. A truncated protein, GBD-C, which models autoinhibited WASP, folds into a highly stable conformation with amide exchange protection factors of up to 3 x 10(6). A novel hydrogen exchange labeling-quench strategy, employing a high-affinity ligand to displace Cdc42 from WASP, was used to examine the amide exchange from the Cdc42-bound state of GBD-C. The GTPase increases exchange rates of the most protected amides by 50-500-fold, with destabilization reducing the differences in the protection of segments in the free state. The results confirm that Cdc42 facilitates the physical separation of the GBD from the VCA in a tethered molecule, indicating this process likely plays an important role in activation of full-length WASP by the GTPase. However, destabilization of GBD-C is not complete in the Cdc42 complex. The data indicate that partitioning of free energy between binding and activation may limit the extent to which GTPases can cause conformational change in effectors. This notion is consistent with the requirement of multiple input signals in order to achieve maximal activation in many effector molecules.

Actins↗
PubMed2004

Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex.

The assembly of a branched network of actin filaments provides the mechanical propulsion that drives a range of dynamic cellular processes, including cell motility. The Arp2/3 complex is a crucial component of such filament networks. Arp2/3 nucleates new actin filaments while bound to existing filaments, thus creating a branched network. In recent years, a number of proteins that activate the filament nucleation activity of Arp2/3 have been identified, most notably the WASP (Wiskott-Aldrich syndrome protein) family. WASP-family proteins activate the Arp2/3 complex, and consequently stimulate actin assembly, in response to extracellular signals. Structural studies have provided a significant refinement in our understanding of the molecular detail of how the Arp2/3 complex nucleates actin filaments. There has also been much progress towards an understanding of the complicated signalling processes that regulate WASP-family proteins. In addition, the use of gene disruption in a number of organisms has led to new insights into the specific functions of individual WASP-family members. The present review will discuss the Arp2/3 complex and its regulators, in particular the WASP-family proteins. Emphasis will be placed on recent developments in the field that have furthered our understanding of actin dynamics and cell motility.

Actin Cytoskeleton↗
PubMed2002

WASp verprolin homology, cofilin homology, and acidic region domain-mediated actin polymerization is required for T cell development.

All members of the Wiskott-Aldrich syndrome protein (WASp) family contain a carboxyl-terminal verprolin homology, cofilin homology, and acidic region (VCA) domain that binds and activates the Arp2/3 complex, thereby linking these proteins to the induction of actin polymerization. Although the VCA domain imbues WASp and other WASp family members with the capacity to modulate cytoskeletal organization, little is known about the impact of this domain activity on lymphoid cell function. Here we demonstrate that T cell-restricted expression of VCA domain-deleted WASp (WASpdeltaVCA) in WAS(-/-) mice engenders a severe early block in T lymphopoiesis associated with impaired T cell antigen receptor alphabeta expression and a consequent failure to generate single-positive CD4(+) and CD8(+) T cells. These latter defects, which are not observed in WAS(-/-) mice, are associated with impaired induction of cellular actin polymerization and a failure in the terminal differentiation of double-negative thymocytes. These findings indicate that WASp family proteins play an essential role in modulating the signaling events required for early thymocyte development and reveal their capacity to subserve this role to depend on VCA domain-mediated actin polymerization.

Actin Depolymerizing Factors↗
PubMed2005

The NF2 tumor suppressor Merlin and the ERM proteins interact with N-WASP and regulate its actin polymerization function.

The function of the NF2 tumor suppressor merlin has remained elusive despite increasing evidence for its role in actin cytoskeleton reorganization. The closely related ERM proteins (ezrin, radixin, and moesin) act as linkers between the cell membrane and cytoskeleton, and have also been implicated as active actin reorganizers. We report here that merlin and the ERMs can interact with and regulate N-WASP, a critical regulator of actin dynamics. Merlin and moesin were found to inhibit N-WASP-mediated actin assembly in vitro, a function that appears independent of their ability to bind actin. Furthermore, exogenous expression of a constitutively active ERM inhibits N-WASP-dependent Shigella tail formation, suggesting that the ERMs may function as inhibitors of N-WASP function in vivo. This novel function of merlin and the ERMs illustrates a mechanism by which these proteins directly exert their effects on actin reorganization and also provides new insight into N-WASP regulation.

Actins↗
PubMed2004

N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor.

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.

Actins↗
PubMed2006

IQGAP1 stimulates actin assembly through the N-WASP-Arp2/3 pathway.

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.

Actin-Related Protein 2↗
PubMed2002

Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP.

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.

Animals↗
PubMed2001

Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells.

In mammalian cells, actin dynamics is tightly controlled through small GTPases of the Rho family, WASP/Scar proteins and the Arp2/3 complex. We employed Cre/loxP-mediated gene targeting to disrupt the ubiquitously expressed N-WASP in the mouse germline, which led to embryonic lethality. To elucidate the role of N-WASP at the cellular level, we immortalized embryonic fibroblasts and selected various N-WASP-defective cell lines. These fibroblasts showed no apparent morphological alterations and were highly responsive to the induction of filopodia, but failed to support the motility of Shigella flexneri. In addition, enteropathogenic Escherichia coli were incapable of inducing the formation of actin pedestals in N-WASP-defective cells. Our results prove the essential role of this protein for actin cytoskeletal changes induced by these bacterial pathogens in vivo and in addition show for the first time that N-WASP is dispensable for filopodia formation.

Actins↗
PubMed1994

A comparative study of IgG subclass antibodies in patients allergic to wasp or bee venom.

IgG1 and IgG4 antivenom antibody responses were compared in groups of patients who had experienced systemic reactions to wasp (Vespula spp.) or bee stings. Pretreatment serum IgG4 antibody levels were low in both groups, but IgG1 antibodies were significantly raised in bee-allergic patients (P < 0.002), probably reflecting their greater exposure to stings than wasp-reactive patients. No direct or indirect relationships were found, in untreated bee or wasp patients, between IgG1, IgG4, or IgE antibody levels and the severity of a patient's last systemic reaction to a sting. After a 12-week course of venom immunotherapy (VIT), IgG1 antibodies increased significantly only in wasp-sensitive patients (P < 0.001), although both groups responded with marked increases in venom-specific IgG4 (P < 0.01). Wasp-allergic subjects who responded to VIT with high production of specific IgG4 showed the greatest increases (pre- to post-VIT) in IgE antibodies (P < 0.05). This group also demonstrated a direct correlation (P < 0.05) between post-VIT levels of IgE and IgG1 antibodies, a finding contrary to an IgE-immunoregulatory role for IgG1. High levels of venom-specific IgG1 alone, or in combination with IgG4, were not protective in three patients who suffered repeated adverse reactions to bee VIT, showing that absolute levels of IgG subclass antivenom antibodies are not reliably indicative of clinical responsiveness in individual patients.

Adult↗
PubMed2003

Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth.

Rho family small GTPases regulate multiple cellular functions through reorganization of the actin cytoskeleton. Among them, Cdc42 and Tc10 induce filopodia or peripheral processes in cultured cells. We have identified a member of the family, designated as RhoT, which is closely related to Tc10. Tc10 was highly expressed in muscular tissues and brain and remarkably induced during differentiation of C2 skeletal muscle cells and neuronal differentiation of PC12 and N1E-115 cells. On the other hand, RhoT was predominantly expressed in heart and uterus and induced during neuronal differentiation of N1E-115 cells. Tc10 exogenously expressed in fibroblasts generated actin-filament-containing peripheral processes longer than the Cdc42-formed filopodia, whereas RhoT produced much longer and thicker processes containing actin filaments. Furthermore, both Tc10 and RhoT induced neurite outgrowth in PC12 and N1E-115 cells, but Cdc42 did not do this by itself. Tc10 and RhoT as well as Cdc42 bound to the N-terminal CRIB-motif-containing portion of N-WASP and activated N-WASP to induce Arp2/3-complex-mediated actin polymerization. The formation of peripheral processes and neurites by Tc10 and RhoT was prevented by the coexpression of dominant-negative mutants of N-WASP. Thus, N-WASP is essential for the process formation and neurite outgrowth induced by Tc10 and RhoT. Neuronal differentiation of PC12 and N1E-115 cells induced by dibutyryl cyclic AMP and by serum starvation, respectively, was prevented by dominant-negative Cdc42, Tc10 and RhoT. Taken together, all these Rho family proteins are required for neuronal differentiation, but they exert their functions differentially in process formation and neurite extension. Consequently, N-WASP activated by these small GTPases mediates neuronal differentiation in addition to its recently identified role in glucose uptake.

3T3 Cells↗
PubMed1997

Variable expression of WASP in B cell lines of Wiskott-Aldrich syndrome patients.

The Wiskott-Aldrich syndrome (WAS) arises from defects of the X-chromosome gene WASP. Severe platelet defects, thrombocytopenia with small platelets, are a hallmark of the disease, but clinical immunodeficiency based in lymphocyte dysfunction varies from negligible to life threatening among WAS patients. To address the connection between WASP mutations and clinical outcomes, we generated and characterized a panel of patient B cell lines. Three cell lines from patients with exon 2 missense mutations and mild immune dysfunction were found to express substantial levels of WASP mRNA and protein. On the other hand, 8 of 10 cell lines from patients with moderate or severe immune dysfunction lack detectable WASP protein. The findings suggest that the clinical variability of the WAS can partially be explained by the level of WASP protein in the patient's cells.

Adolescent↗