scholarly journals SCAR knockouts in Dictyostelium: WASP assumes SCAR’s position and upstream regulators in pseudopods

2012 ◽  
Vol 198 (4) ◽  
pp. 501-508 ◽  
Author(s):  
Douwe M. Veltman ◽  
Jason S. King ◽  
Laura M. Machesky ◽  
Robert H. Insall
Keyword(s):  
Dictyostelium Discoideum ◽  
Traveling Waves ◽  
Actin Polymerization ◽  
Leading Edge ◽  
The Other ◽  
Coated Pits ◽  
Wiskott Aldrich Syndrome ◽  
Regulatory Complex ◽  
Syndrome Protein ◽  
Upstream Regulators

Under normal conditions, the Arp2/3 complex activator SCAR/WAVE controls actin polymerization in pseudopods, whereas Wiskott–Aldrich syndrome protein (WASP) assembles actin at clathrin-coated pits. We show that, unexpectedly, Dictyostelium discoideum SCAR knockouts could still spread, migrate, and chemotax using pseudopods driven by the Arp2/3 complex. In the absence of SCAR, some WASP relocated from the coated pits to the leading edge, where it behaved with similar dynamics to normal SCAR, forming split pseudopods and traveling waves. Pseudopods colocalized with active Rac, whether driven by WASP or SCAR, though Rac was activated to a higher level in SCAR mutants. Members of the SCAR regulatory complex, in particular PIR121, were not required for WASP regulation. We thus show that WASP is able to respond to all core upstream signals and that regulators coupled through the other members of SCAR’s regulatory complex are not essential for pseudopod formation. We conclude that WASP and SCAR can regulate pseudopod actin using similar mechanisms.

scholarly journals ADictyosteliumHomologue of WASP Is Required for Polarized F-Actin Assembly during Chemotaxis

2005 ◽  
Vol 16 (5) ◽  
pp. 2191-2206 ◽  
Author(s):  
Scott A. Myers ◽  
Ji W. Han ◽  
Yoonsung Lee ◽  
Richard A. Firtel ◽  
Chang Y. Chung
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Essential Role ◽  
Leading Edge ◽  
Actin Assembly ◽  
Temporal Control ◽  
Wiskott Aldrich Syndrome ◽  
Low Levels ◽  
Syndrome Protein

The actin cytoskeleton controls the overall structure of cells and is highly polarized in chemotaxing cells, with F-actin assembled predominantly in the anterior leading edge and to a lesser degree in the cell's posterior. Wiskott-Aldrich syndrome protein (WASP) has emerged as a central player in controlling actin polymerization. We have investigated WASP function and its regulation in chemotaxing Dictyostelium cells and demonstrated the specific and essential role of WASP in organizing polarized F-actin assembly in chemotaxing cells. Cells expressing very low levels of WASP show reduced F-actin levels and significant defects in polarized F-actin assembly, resulting in an inability to establish axial polarity during chemotaxis. GFP-WASP preferentially localizes at the leading edge and uropod of chemotaxing cells and the B domain of WASP is required for the localization of WASP. We demonstrated that the B domain binds to PI(4,5)P2and PI(3,4,5)P3with similar affinities. The interaction between the B domain and PI(3,4,5)P3plays an important role for the localization of WASP to the leading edge in chemotaxing cells. Our results suggest that the spatial and temporal control of WASP localization and activation is essential for the regulation of directional motility.

scholarly journals Fyn and PTP-PEST–mediated Regulation of Wiskott-Aldrich Syndrome Protein (WASp) Tyrosine Phosphorylation Is Required for Coupling T Cell Antigen Receptor Engagement to WASp Effector Function and T Cell Activation

2004 ◽  
Vol 199 (1) ◽  
pp. 99-112 ◽  
Author(s):  
Karen Badour ◽  
Jinyi Zhang ◽  
Fabio Shi ◽  
Yan Leng ◽  
Michael Collins ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Actin Polymerization ◽  
Cell Activation ◽  
Synapse Formation ◽  
Tyrosine Phosphatase ◽  
Structural Constraints ◽  
Wiskott Aldrich Syndrome ◽  
Syndrome Protein

Involvement of the Wiskott-Aldrich syndrome protein (WASp) in promoting cell activation requires its release from autoinhibitory structural constraints and has been attributed to WASp association with activated cdc42. Here, however, we show that T cell development and T cell receptor (TCR)-induced proliferation and actin polymerization proceed normally in WASp−/− mice expressing a WASp transgene lacking the cdc42 binding domain. By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation. TCR-induced WASp tyrosine phosphorylation was also disrupted in T cells lacking Fyn, a kinase shown here to bind, colocalize with, and phosphorylate WASp. By contrast, WASp was tyrosine dephosphorylated by protein tyrosine phosphatase (PTP)-PEST, a tyrosine phosphatase shown here to interact with WASp via proline, serine, threonine phosphatase interacting protein (PSTPIP)1 binding. Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation. These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.

scholarly journals TheSaccharomyces cerevisiaeHomologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex

1999 ◽  
Vol 10 (10) ◽  
pp. 3521-3538 ◽  
Author(s):  
Ammar Madania ◽  
Pascal Dumoulin ◽  
Sandrine Grava ◽  
Hiroko Kitamoto ◽  
Claudia Schärer-Brodbeck ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Temperature Sensitive ◽  
Multicopy Suppressor ◽  
Wiskott Aldrich Syndrome ◽  
Regulatory Cascades ◽  
Src Homology ◽  
Synthetically Lethal ◽  
Actin Patch ◽  
Syndrome Protein

Yeast Las17 protein is homologous to the Wiskott–Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least oneARP3 ts mutant are synthetically lethal withlas17Δ ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Δcells showed that receptor-mediated internalization of α factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.

Tyrosine Phosphorylation of WASP Promotes Calpain-Mediated Podosome Disassembly In Myeloid Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1498-1498
Author(s):  
Lee Macpherson ◽  
James Monypenny ◽  
Michael P Blundell ◽  
Giles O Cory ◽  
Jessica Tomé-García ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Actin Polymerization ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Reference List ◽  
Wiskott Aldrich Syndrome ◽  
Open Conformation ◽  
New Findings ◽  
Syndrome Protein

Abstract Abstract 1498 We have previously shown that a point mutation (Ile294Thr) in the Wiskott Aldrich Syndrome Protein (WASP) detected in a Wiskott Aldrich Syndrome (WAS) patient led to enhanced actin polymerising activity of WASP 1 and well as increased instability2. We also showed that macrophages from this patient displayed an increased number of actin based adhesion structures called podosomes. Additionally, podosomes in macrophages from this patient were extremely dynamic with a high rate of turnover. Based on these results we proposed that the active open conformation of WASP promotes actin polymerisation but it also induces podosome disassembly and adhesion turnover. Although the idea that the same open conformation of WASP leads to both assembly and disassembly of podosomes may be counterintuitive at first, it is possible that for termination of podosomes actin polymerisation and integrin recruitment have to be discontinued and the same constituents of growing podosomes such as active WASP may contribute to the disassembly process. We have also shown that the rapid turn-over of podosomes involves cleavage of WASP by the protease calpain, further supporting a role of WASP in both podosome formation and disassembly. However, the specific signalling mechanisms that make active WASP susceptible to cleavage by calpain leading to podosome disassembly remain unknown and need further clarification. Phosphorylation of WASP Y291 (human) or Y293 (mouse) promotes the open conformation of WASP and results in enhanced actin polymerisation. We now report that in myeloid cells, tyrosine phosphorylation of WASP negatively regulates the stability of podosomes, leading to their calpain-dependent disassembly. Additionally, we found that constitutive phosphorylation of WASP results in extensive degradation in a process that involves calpain. Our data also indicate that phosphorylation of WASP sustains the open/active conformation that promotes cleavage of WASP by calpain. Interestingly, phosphorylated WASP can bind to the WASP interacting protein (WIP) and other proteins that form a complex with WASP in podosomes such as Nck, cortactin. Taken together, our data indicate that in myeloid cells, tyrosine phosphorylation sustains the open conformation of WASP and it enhances its susceptibility to calpain-mediated cleavage preventing accumulation of actin filaments and integrin associated proteins in podosomes. This process facilitates podosome disassembly and cell translocation. These new findings support the key role of WASP as a protein that integrates actin polymerisation and cell adhesion required for mobilisation of myeloid cells during the immune response. Reference List 1. Ancliff PJ, Blundell MP, Cory GO et al. Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia. Blood 2006;108:2182-2189. 2. Moulding DA, Blundell MP, Spiller DG et al. Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia. J Exp. Med 2007;204:2213–2224. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cryptosporidium parvum Infection Requires Host Cell Actin Polymerization

2001 ◽  
Vol 69 (9) ◽  
pp. 5940-5942 ◽  
Author(s):  
David A. Elliott ◽  
Daniel J. Coleman ◽  
Michael A. Lane ◽  
Robin C. May ◽  
Laura M. Machesky ◽  
...  
Keyword(s):  
Host Cell ◽  
Cryptosporidium Parvum ◽  
Actin Polymerization ◽  
Protozoan Parasite ◽  
Parasite Infection ◽  
Cell Cytoplasm ◽  
Wiskott Aldrich Syndrome ◽  
Host Cell Cytoplasm ◽  
Syndrome Protein ◽  
Intracellular Protozoan Parasite

ABSTRACT The intracellular protozoan parasite Cryptosporidium parvum accumulates host cell actin at the interface between the parasite and the host cell cytoplasm. Here we show that the actin polymerizing proteins Arp2/3, vasodilator-stimulated phosphoprotein (VASP), and neural Wiskott Aldrich syndrome protein (N-WASP) are present at this interface and that host cell actin polymerization is necessary for parasite infection.

scholarly journals The Intersectin 2 Adaptor Links Wiskott Aldrich Syndrome Protein (WASp)-mediated Actin Polymerization to T Cell Antigen Receptor Endocytosis

2001 ◽  
Vol 194 (12) ◽  
pp. 1777-1787 ◽  
Author(s):  
Mary K.H. McGavin ◽  
Karen Badour ◽  
Lynne A. Hardy ◽  
Terrance J. Kubiseski ◽  
Jinyi Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Antigen Receptor ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Wiskott Aldrich Syndrome ◽  
Syndrome Protein ◽  
Dh Domain

Induction of T cell antigen receptor (TCR) endocytosis has a significant impact on TCR signaling and T cell behavior, but the molecular interactions coordinating internalization of the activated TCR are poorly understood. Previously we have shown that TCR endocytosis is regulated by the Wiskott Aldrich Syndrome protein (WASp), a cytosolic effector which, upon interaction with the cdc42 Rho GTPase, couples TCR engagement to Arp 2/3 complex-mediated actin polymerization. Here we report that WASp associates in T cells with intersectin 2, an endocytic adaptor containing multiple domains including a Dbl homology (DH) domain with the potential to activate Rho GTPases. Intersectin 2 association with WASp increases after TCR engagement, and its overexpression in Cos-7 cells induces WASp translocation to endocytic vesicles within which intersectin 2 colocalizes with both WASp and cdc42. Intersectin 2, but not a DH domain-deleted (ΔDH) form of intersectin 2, and stimulation via the TCR also trigger the activation of cdc42. Induction of TCR internalization is also augmented by intersectin 2 and severely impaired by latrunculin B treatment. Thus, intersection 2 appears to function cooperatively with WASp and cdc42 to link the clathrin endocytic machinery to WASp-mediated actin polymerization and ultimately to occupancy-induced TCR endocytosis.

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