Timothy S. Gomez

A FAM21-Containing WASH Complex Regulates Retromer-Dependent Sorting

The Arp2/3 complex regulates endocytosis, sorting, and trafficking, yet the Arp2/3-stimulating factors orchestrating these distinct events remain ill defined. WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homolog) is an Arp2/3 activator with unknown function that was duplicated during primate evolution. We demonstrate that WASH associates with tubulin and localizes to early endosomal subdomains, which are enriched in Arp2/3, F-actin, and retromer components. Although WASH localized with activated receptors, it was not essential for endocytosis. However, WASH did regulate retromer-mediated retrograde CI-MPR trafficking, which required its association with endosomes, Arp2/3-directed F-actin regulation, and tubulin interaction. Moreover, WASH exists in a multiprotein complex containing FAM21, which links WASH to endosomes and is required for WASH-dependent retromer-mediated sorting. Significantly, without WASH, retromer tubulation was exaggerated, supporting a model wherein WASH links retromer-mediated cargo containing tubules to microtubules for Golgi-directed trafficking and generates F-actin-driven force for tubule scission.

Regulation of T-cell activation by the cytoskeleton

To become activated, T cells must efficiently recognize antigen-presenting cells or target cells through several complex cytoskeleton-dependent processes, including integrin-mediated adhesion, immunological-synapse formation, cellular polarization, receptor sequestration and signalling. The actin and microtubule systems provide the dynamic cellular framework that is required to orchestrate these processes and ultimately contol T-cell activation. Here, we discuss recent advances that have furthered our understanding of the crucial importance of the T-cell cytoskeleton in controlling these aspects of T-cell immune recognition.

Structure and control of the actin regulatory WAVE complex.

Members of the Wiskott–Aldrich syndrome protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation with the Arp2/3 complex. The WASP relative WAVE regulates lamellipodia formation within a 400-kilodalton, hetero-pentameric WAVE regulatory complex (WRC). The WRC is inactive towards the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. Here we report the 2.3-ångstrom crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting the way in which these signals stimulate WRC activity towards the Arp2/3 complex. The spatial proximity of the Rac binding site and the large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.

Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis

The absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)(2) domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (deltapsi(m)), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP.

The WAVE2 Complex Regulates Actin Cytoskeletal Reorganization and CRAC-Mediated Calcium Entry during T Cell Activation

BACKGROUND The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. RESULTS By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. CONCLUSIONS These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

G Protein-Coupled Chemokine Receptors Induce Both Survival and Apoptotic Signaling Pathways

Chemokine receptors are essential for triggering chemotaxis to immune cells; however, a number of them can also mediate death when engaged by nonchemokine ligands. When the chemokine receptor CXCR4 is engaged by stromal cell-derived factor (SDF1)α, it triggers cells to chemotax, and in some cell types such as neurons, causes cell death. To elucidate this dual and opposing receptor function, we have investigated whether CXCR4 activation by its chemokine SDF1α could lead to the simultaneous activation of both anti- and proapoptotic signaling pathways; the balance ultimately influencing cell survival. CXCR4 activation in CD4 T cells by SDF1α led to the activation of the prosurvival second messengers, Akt and extracellular signal-regulated protein kinase. Selective inhibition of each signal demonstrated that extracellular signal-regulated protein kinase is essential for mediating SDF1α-triggered chemotaxis but does not confer an antiapoptotic state. In contrast, Akt activation through CXCR4 by SDF1α interactions is necessary to confer resistance to apoptosis. The proapoptotic signaling pathway triggered by SDF1α-CXCR4 interaction involves the Giα protein-independent activation of the proapoptotic MAPK (p38). Furthermore, other chemokines and chemokine receptors also signal chemotaxis and proapoptotic effects via similar pathways. Thus, Giα protein-coupled chemokine receptors can function as death prone receptors and the balance between the above signaling pathways will ultimately mandate the fate of the activated cell.

Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse

Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor–stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.

WASH and WAVE actin regulators of the Wiskott–Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes

We recently showed that the Wiskott–Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

T cell activation and the cytoskeleton: you can't have one without the other.

More than a quarter of a century has passed since the observation that T cells rapidly polarize their actin and microtubule cytoskeletal systems toward antigen-presenting cells during activation. Since this initial discovery, several receptors on T cells (e.g., T cell receptor [TCR], co-receptors, integrins, and chemokine receptors) have been identified to regulate these two cytoskeletal networks through complex signaling pathways, which are still being elucidated. There is now an undeniable body of biochemical, pharmacological, and genetic evidence indicating that regulators of actin and microtubule dynamics are crucial for T cell activation and effector functions. In fact, the actin cytoskeleton participates in the initial clustering of TCR-major histocompatibility complex or peptide complexes, formation and stabilization of the immune synapse, integrin-mediated adhesion, and receptor sequestration, whereas both the actin and microtubule cytoskeletons regulate the establishment of cell polarity, cell migration, and directed secretion of cytokines and cytolytic granules. Over the past several years, we have begun to more thoroughly understand the contributions of specific actin-regulatory and actin-nucleating proteins that govern these processes. Herein, we discuss our current understanding of how activating receptors on T lymphocytes regulate the actin and microtubule cytoskeletons, and how in turn, these distinct but integrated cytoskeletal networks coordinate T cell immune responses.

Human Immunodeficiency Virus–Induced Apoptosis of Human Hepatocytes via CXCR4

Human immunodeficiency virus (HIV) infection is commonly associated with liver dysfunction. The X4 HIV glycoprotein 120 envelope (env) induces apoptosis in T cells and neurons via the HIV coreceptor CXCR4. Therefore, we investigated whether hepatocyte death could result from the HIV env signaling through CXCR4 on the hepatocyte. We demonstrated that hepatocytes in humans express CXCR4 on the cell surface. Furthermore, we established that the X4 HIV env and the entire HIV virion signal hepatocyte apoptosis through CXCR4. The apoptotic process is dependent on G(ialpha) protein signaling, yet it is independent of caspase cascade activation. Thus, HIV can directly cause hepatocyte death in humans by signaling through CXCR4, without infecting the cell.