Search

The Wiskott-Aldrich syndrome (WAS) interacting protein (WIP) stabilizes the WAS protein (WASP), the product of the gene mutated in WAS. WIP-deficient T cells have low WASP levels, limiting the usefulness of WIP KO mice in defining the role of WIP in T cell function. To define this role, we compared WIP/WASP double KO (DKO) mice to WASP KO mice on DO11.10 background. T cell development was normal in both strains, but peripheral T cell numbers were significantly decreased in DKO mice. WASP KO T cells proliferated and secreted IL-2 normally in response to OVA peptide (OVAp). In contrast, T cells from DKO mice proliferated poorly in response to OVAp in vitro, and cutaneous hapten hypersensitivity was deficient in these mice. DKO T cells up-regulated CD25 expression and secreted normal amounts of IL-2 after antigen stimulation, but had defective response to IL-2, evidenced by failure to further up-regulate CD25 expression, phosphorylate STAT5, and induce expression of STAT5-dependent genes. DKO, but not WASP KO, T cells had a disrupted subcortical actin cytoskeleton and impaired actin polymerization after T cell antigen receptor (TCR) ligation. These results indicate that WIP is essential for IL-2 signaling and responsiveness in T cells, possibly because of its critical role in TCR-triggered actin cytoskeletal reorganization.

Neutrophil chemotaxis depends on actin dynamics, but the roles for specific cytoskeleton regulators in this response remain unclear. By analysis of mammalian diaphanous-related formin 1 (mDia1)-deficient mice, we have identified an essential role for this actin nucleator in neutrophil chemotaxis. Lack of mDia1 was associated with defects in chemoattractant-induced neutrophil actin polymerization, polarization, and directional migration, and also with impaired activation of RhoA, its downstream target p160-Rho-associated coil-containing protein kinase (ROCK), and the leukemia-associated RhoA guanine nucleotide exchange factor (LARG). Our data also revealed mDia1 to be associated with another cytoskeletal regulator, Wiskott-Aldrich syndrome protein (WASp), at the leading edge of chemotaxing neutrophils and revealed polarized morphology and chemotaxis to be more mildly impaired in WAS(-/-) than in mDia1(-/-) neutrophils, but essentially abrogated by combined mDia1/WASp deficiency. Thus, mDia1 roles in neutrophil chemotaxis appear to be subserved in concert with WASp and are realized at least in part by activation of the LARG/RhoA/ROCK signaling pathway.

Wiskott-Aldrich syndrome is caused by alterations in the Wiskott-Aldrich syndrome protein (WASP) and several of these mutations affect WASP's interaction with WIP (WASP-interacting protein), suggesting that loss of interaction between WASP and WIP is causal to the disease. Las17p is the yeast homologue of WASP and las17Delta strain is unable to grow at 37 degrees C. We show that Human WASP suppresses the growth defect of Saccharomyces cerevisiae las17Delta strain, only in the presence of WIP. WIP mediates cortical localisation of WASP as well as stabilise WASP in yeast cells. Mutations which affected WASP-WIP interaction abolished WASP's ability to suppress the growth defect of las17Delta strain. We have demonstrated that WASP-WIP is an active complex and WASP's ability to suppress the growth defect of las17Delta strain is dependent on the presence of a functional Arp2/3 activating domain of WASP and also the Verprolin domain (V) of WIP.

Homing of lymphocytes to tissues is a biologically important multistep process that involves selectin-dependent rolling, integrin-dependent adhesion and chemokine-directed chemotaxis. The actin cytoskeleton plays a central role in lymphocyte adhesion and motility. Wiskott-Aldrich syndrome protein (WASP), the product of the gene mutated in Wiskott-Aldrich syndrome, and its partner, the Wiskott-Aldrich syndrome protein-interacting protein (WIP), play important roles in actin re-organization in T lymphocytes. We used mice with disruption of the WASP and WIP genes to examine the role of WASP and WIP in T cell homing. T cell homing to spleen and lymph nodes in vivo was deficient in WASP-/- and WIP-/- mice and severely impaired in WASP-/-WIP-/- double knockout (DKO) mice. Deficiency of WASP, WIP or both did not interfere with selectin-dependent rolling or integrin-dependent adhesion of T cells in vitro. Chemotaxis to stromal cell-derived factor-1alpha (SDF-1alpha) in vitro was mildly reduced in T cells from WASP-/- mice. In contrast, it was significantly impaired in T cells from WIP-/- mice and severely reduced in T cells from DKO mice. Cellular F-actin increase following SDF-1alpha stimulation was normal in WASP-/- and WIP-/- T cells, but severely reduced in T cells from DKO mice. Actin re-organization and polarization in response to SDF-1alpha was abnormal in T cells from all knockout mice. Early biochemical events following SDF-1alpha stimulation that are important for chemotaxis and that included phosphorylation of Lck, cofilin, PAK1 and extracellular regulated kinase (Erk) and GTP loading of Rac-1 were examined in T cells from DKO mice and found to be normal. These results suggest that WASP and WIP are not essential for T lymphocyte rolling and adhesion, but play important and partially redundant roles in T cell chemotaxis in vitro and homing in vivo and function downstream of small GTPases.

The importance of coupling the process of endocytosis to factors that regulate actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified. Sla1p is a well-characterized yeast protein that binds both to activators of actin dynamics, Las17p and Pan1p, and to cargo proteins, such as the pheromone receptor Ste2p. Previously, we reported that the Lsb5 protein plays a role in endocytosis in yeast and that it localizes to the plasma membrane. Lsb5p has a similar structure to the GGA [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS [Vps27p (vacuolar protein sorting protein 27), Hrs, Stam] domain and a GAT (GGA and Tom1) domain. It does not, however, contain either a gamma-adaptin ear or a clathrin-binding motif. In the present study, we have further defined its interaction site with both Sla1p and with Las17p, two regulators of actin dynamics. The site of interaction with Sla1p involves the Sla1 HD1 (homology domain 1), which also was shown previously to interact with the pheromone receptor Ste2p. We also demonstrate hitherto unknown interactions between Lsb5p and the active form of the yeast Arf3 protein, and with ubiquitin. Finally, we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the correct cortical site in cells. Taken together, our data provide further evidence for the role of Lsb5p in membrane-trafficking events at the plasma membrane and also demonstrate for the first time an interaction of Arf3 with the endocytic machinery in yeast.