Andes Virus Disrupts the Endothelial Cell Barrier by Induction of Vascular Endothelial Growth Factor and Downregulation of VE-Cadherin

Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS) are severe diseases associated with hantavirus infection. High levels of virus replication occur in microvascular endothelial cells but without a virus-induced cytopathic effect. However, virus infection results in microvascular leakage, which is the hallmark of these diseases. VE-cadherin is a major component of adherens junctions, and its interaction with the vascular endothelial growth factor (VEGF) receptor, VEGF-R2, is important for maintaining the integrity of the endothelial barrier. Here we report that increased secreted VEGF and concomitant decreased VE-cadherin are seen at early times postinfection of human primary lung endothelial cells with an HPS-associated hantavirus, Andes virus. Furthermore, active virus replication results in increased permeability and loss of the integrity of the endothelial cell barrier. VEGF binding to VEGF-R2 is known to result in dissociation of VEGF-R2 from VE-cadherin and in VE-cadherin activation, internalization, and degradation. Consistent with this, we showed that an antibody which blocks VEGF-R2 activation resulted in inhibition of the Andes virus-induced VE-cadherin reduction. These data implicate virus induction of VEGF and reduction in VE-cadherin in the endothelial cell permeability seen in HPS and suggest potential immunotherapeutic targets for the treatment of the disease.Hantaviruses, of the family Bunyaviridae, are rodent-borne RNA viruses. Members of the Hantavirus genus have been identified as etiologic agents of two severe human diseases: hemorrhagic fever with renal syndrome (HFRS), which is caused by the Old World hantaviruses, and hantavirus pulmonary syndrome (HPS), which is caused by the New World hantaviruses (38, 39). Sin Nombre virus (SNV) and Andes virus (ANDV) are the main causes of HPS in the Americas. The major hantavirus target in humans is the microvascular endothelium, and the basis of HPS and HFRS is attributed to microvascular leakage (9, 34, 57). Common clinical features of HPS are interstitial pneumonitis with variable amounts of mononuclear cell infiltration, congestion, and both interstitial and alveolar edema (4, 34, 57). Despite the prominent accumulation of viral antigen in the infected vascular endothelium, no evidence of cellular destruction has been observed (57). Absence of a cytopathic effect has also been reported in in vitro studies of hantavirus infection of human primary endothelial cells (35, 46). In general, it is believed that induction of an uncontrolled immune response to the hantavirus infection, rather than the viral infection per se, is the cause of the microvascular leakage and ultimately HPS and HFRS (3, 48, 57). So far, a limited number of in vitro permeability studies have reported either no significant changes in the vascular permeability upon hantavirus infection or a significant increase only when mediators of increased permeability are exogenously added to the hantavirus-infected cells (12, 22, 46).Endothelial cell permeability is a highly regulated process and is maintained by both tight and adherens junctions (47). The disruption of adherens junctions is sufficient to disturb the endothelium barrier function and cause an increase in permeability and formation of edema (25, 47). Adherens junctions are largely composed of vascular endothelial (VE) cadherin (VE-cadherin), an endothelial cell-specific member of the cadherin family of adhesion protein (51, 52). Adherens junctions and in particular VE-cadherin are targets of the signaling pathway of agents that increase vascular permeability (7, 8, 10). Vascular endothelial growth factor (VEGF), one of the most potent vascular permeability agents, exerts its effects after binding to its homologous membrane tyrosine kinase receptor, VEGF-R2, whose expression is restricted to endothelial cells. It is known that VEGF-R2 interacts with VE-cadherin, and together they maintain the endothelial cell barrier (26). When VEGF is present, it binds to VEGF-R2, and that initiates the internalization and degradation of VE-cadherin and disruption of the adherens junctions (10, 54).In general, increase of vascular permeability is an important component of severe disease progression in hemorrhagic fevers (36). A number of studies have investigated the cause of increased vascular permeability in viral hemorrhagic fevers induced by viruses such as Dengue virus or Ebola virus (41, 42, 50, 53, 56). Studies of vascular permeability during hantavirus infection in vitro have mainly been performed in the presence of various inflammatory agents and growth factors (12, 15, 19, 22, 46). A recent study demonstrated that pathogenic hantaviruses sensitize the endothelium and cause hyperpermeability in response to high levels of exogenously added VEGF (12). We show here that VE-cadherin downregulation can be observed in ANDV-infected cells in the absence of exogenous VEGF. The downregulation of VE-cadherin in the absence of exogenous VEGF led us to the discovery that endothelial cells infected with ANDV induce the production of VEGF at early times postinfection. The early increased secretion of VEGF coincides with the initiation of downregulation of the adherent junction protein VE-cadherin and an increase in permeability of endothelial cells. The involvement of VEGF-R2 in VE-cadherin downregulation was demonstrated by antibody blockage of VEGF-R2 that resulted in significant recovery of VE-cadherin levels. These data indicate that the increased vascular permeability seen in HPS could be a direct result of hantavirus infection of the endothelium and may occur through a pathway involving VEGF-induced downregulation of VE-cadherin at early times postinfection.     

Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization

Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.The ability of a cell to rapidly respond to extracellular cues and direct cytoskeletal rearrangements is dependent on an array of signaling complexes that control actin assembly (58). The protrusive structures at the leading edges of motile cells are broadly defined as lamellipodia or filopodia (14). Lamellae are sheet-like protrusions composed of dendritic actin arrays that drive membrane expansion, with the “lamellipodium” representing a narrow region at the edge of the cell (in culture) characterized by rapid actin polymerization. This F-actin assembly is suggested to require Arp2/3 activity that nucleates new actin filaments from the sides of existing ones (58, 71) and capping proteins that limit the length of these new filaments and stabilize them (7). Arp2/3 activity in turn is regulated by the WASP/WAVE family of proteins, such as N-WASP and WAVE2 (68), whose regulation is a subject of intense interest (12, 29, 36, 41, 56, 76).Filopodia contain parallel bundles of actin filaments containing fascin (22). These are dynamic structures that emanate from the periphery of the cell and are retracted, with occasional attachment (to the dish in culture). Thus, they have been thought to have a sensory or exploratory role during cell migration (28). This is the case for neuronal growth cones, where filopodia sense attractant or repulsive cues and dictate direction in axonal path finding (9, 17, 25, 35). Filopodia have been shown to be important in the context of dendritic-spine development (64, 77), epithelial-sheet closure (26, 60, 79), and cell invasion/metastasis (80, 83).Lamellipodia have been well characterized since the pioneering work of Abercrombie et al. in the early 1970s (2, 3, 4). Filopodia require symmetry breaking at the leading edge (initiation), followed by elongation driven by a filopodial-tip protein complex (14, 28). A few proteins have been identified in this complex; Mena/Vasp serve to prevent capping at the barbed ends of bundled actin filaments (7, 53), and Dia2 promotes F-actin elongation (57, 85). Termination of filopodial elongation is not understood but nonetheless is likely to be tightly regulated. In the absence of F-actin elongation, retraction of the filopodium takes place by a rearward flow of F-actin and filament depolymerization (22).IRSp53 is in a position to play a pivotal role in generating filopodia; this brain-enriched protein was discovered as a substrate of the insulin receptor (87). Subsequently, IRSp53 was identified as an effector for Rac1 (50) and Cdc42 (27, 38), where it participates in filopodium and lamellipodium production (38, 51, 54, 86), neurite extension (27), dendritic-spine morphogenesis (1, 15, 66, 67), cell motility and invasiveness (24). The N terminus of IRSp53 contains a conserved helical domain that is found in five different gene products and is referred to as the IRSp53/MIM homology domain (IMD) (51, 70). This domain has been postulated to bind to Rac1 (50, 70) in a nucleotide-independent manner (52), but no convincing effector-like region has been identified. A Cdc42-specific CRIB-like sequence that does not bind Rac1 (27, 38) allows coupling of this and perhaps related Rho GTPases. The structure of the IMD reveals a zeppelin-shaped dimer that could bind “bent” membranes; thus, its potential as an F-actin-bundling domain (51, 82) could be an in vitro artifact often attributed to proteins with basic patches (46). Although there are reports of F-actin binding at physiological ionic strength (ca. 100 mM KCl) (82, 19), this region when expressed in isolation does not decorate F-actin in vivo.Two reports showed the IMD to be an “inverse-BAR” domain. BAR (Bin-amphipysin-Rvs) domains are found in proteins involved in endocytic trafficking, such as amphipysin and endophilin, and stabilize positively bent membranes, such as those on endocytic vesicles (31, 47). The IMD domains of both IRSp53 (70) and MIM-B (46) associate with lipids and can induce tubulations of PI(3,4,5)P₃ or PI(4,5)P₂-rich membranes, respectively. These tubulations are equivalent to membrane protrusions and are also referred to as negatively bent membranes. Ectopic expression of the IMD from IRSp53 (51, 70, 82, 86) or two other family members, MIM-B (11, 46) and IRTKS (52), can give rise to cells with many peripheral extensions. MIM-B is said to stimulate lamellipodia (11), while IRTKS generates “short actin clusters” at the cell periphery (52).In IRSp53 is a CRIB-like motif that mediates binding to Cdc42 (27, 38), but the function of this interaction in unclear. Cdc42 could relieve IRSp53 autoinhibition as described for N-Wasp (38), but there is little evidence for this. It has been suggested that Cdc42 controls IRSp53 localization and actin remodeling (27, 38), but another study indicated that these events are Cdc42 independent (19). IRSp53 contains a central SH3 domain that may bind proline-rich proteins, such as Dia1 (23), Mena (38), WAVE2 (49, 50, 69), and Eps8 (19, 24). However, it seems unlikely that all of these represent bona fide partners, and side-by-side comparison is provided in this study. Mena is involved in filopodium production (37), Dia1 in stress fiber formation (81), and WAVE2 in lamellipodium extension (72). Thus, Mena is a better candidate as a partner for IRSp53-mediated filopodia than Dia1 or WAVE2.There is good evidence for IRSp53 as a cellular partner for Eps8 (19). Eps8 is an adaptor protein containing an N-terminal PTB domain that can associate with receptor tyrosine kinases (65), and perhaps β integrins (13), and a C-terminal SH3 domain that can associate with Abi1 (30). Binding of the general adaptor Abi1 appears to positively regulate the actin-capping domain at the C terminus of Eps8 (18). It has been suggested that IRSp53 and Eps8 as a complex regulate cell motility, and perhaps Rac1 activation, via SOS (24); more recently, their roles in filopodium formation have been addressed (19). The involvement of IRSp53, but not MIM-B or IRTKS, in filopodium formation might be related to its role as a Cdc42 effector. We show here that, surprisingly, the CRIB motif is not essential for this activity, but rather, the ability of IRSp53 to associate via its SH3 domain is required, and that this domain is controlled by 14-3-3 binding.We have focused on the regulation of Cdc42 effectors that bind 14-3-3, including IRSp53 and PAK4, which are found as 14-3-3 targets in various proteomic projects (32, 44). In this study, we characterize the binding of 14-3-3 to IRSp53 and uncover how this activity regulates IRSp53 function. The phosphorylation-dependent 14-3-3 binding is GSK3β dependent, and 14-3-3 blocks the accessibility of both the CRIB and SH3 domains of IRSp53, thus indicating its primary function in controlling IRSp53 partners. This regulation of the SH3 domain by 14-3-3 is critical in the proper localization and termination of IRSp53 function to promote filopodium dynamics.     

Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.Hantaviruses cause 2 human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (50). HPS and HFRS are multifactorial in nature and cause thrombocytopenia, immune and endothelial cell responses, and hypoxia, which contribute to disease (7, 11, 31, 42, 62). Although these syndromes sound quite different, they share common components which involve the ability of hantaviruses to infect endothelial cells and induce capillary permeability. Edema, which results from capillary leakage of fluid into tissues and organs, is a common finding in both HPS and HFRS patients (4, 7, 11, 31, 42, 62). In fact, both diseases can present with renal or pulmonary sequelae, and the renal or pulmonary focus of hantavirus diseases is likely to result from hantavirus infection of endothelial cells within vast glomerular and pulmonary capillary beds (4, 7, 11, 31, 42, 62). All hantaviruses predominantly infect endothelial cells which line capillaries (31, 42, 44, 61, 62), and endothelial cells have a primary role in maintaining fluid barrier functions of the vasculature (1, 12, 55). Although hantaviruses do not lyse endothelial cells (44, 61), this primary cellular target underlies hantavirus-induced changes in capillary integrity. As a result, understanding altered endothelial cell responses following hantavirus infection is fundamental to defining the mechanism of permeability induced by pathogenic hantaviruses (1, 12, 55).Pathogenic, but not nonpathogenic, hantaviruses use β₃ integrins on the surface of endothelial cells and platelets for attachment (19, 21, 23, 39, 46), and β₃ integrins play prominent roles in regulating vascular integrity (3, 6, 8, 24, 48). Pathogenic hantaviruses bind to basal, inactive conformations of β₃ integrins (35, 46, 53) and days after infection inhibit β₃ integrin-directed endothelial cell migration (20, 46). This may be the result of cell-associated virus (19, 20, 22) which keeps β₃ in an inactive state but could also occur through additional regulatory processes that have yet to be defined. Interestingly, the nonpathogenic hantaviruses Prospect Hill virus (PHV) and Tula virus (TULV) fail to alter β₃ integrin functions, and their entry is consistent with the use of discrete α₅β₁ integrins (21, 23, 36).On endothelial cells, α_vβ₃ integrins normally regulate permeabilizing effects of vascular endothelial growth factor receptor-2 (VEGFR2) (3, 24, 48, 51). VEGF was initially identified as an edema-causing vascular permeability factor (VPF) that is 50,000 times more potent than histamine in directing fluid across capillaries (12, 14). VEGF is responsible for disassembling adherens junctions between endothelial cells to permit cellular movement, wound repair, and angiogenesis (8, 10, 12, 13, 17, 26, 57). Extracellular domains of β₃ integrins and VEGFR2 reportedly form a coprecipitable complex (3), and knocking out β₃ causes capillary permeability that is augmented by VEGF addition (24, 47, 48). Pathogenic hantaviruses inhibit β₃ integrin functions days after infection and similarly enhance the permeability of endothelial cells in response to VEGF (22).Adherens junctions form the primary fluid barrier of endothelial cells, and VEGFR2 responses control adherens junction disassembly (10, 17, 34, 57, 63). Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific adherens junction protein and the primary determinant of paracellular permeability within the vascular endothelium (30, 33, 34). Activation of VEGFR2, another endothelial cell-specific protein, triggers signaling responses resulting in VE-cadherin disassembly and endocytosis, which increases the permeability of endothelial cell junctions (10, 12, 17, 34). VEGF is induced by hypoxic conditions and released by endothelial cells, platelets, and immune cells (2, 15, 38, 52). VEGF acts locally on endothelial cells through the autocrine or paracrine activation of VEGFR2, and the disassembly of endothelial cell adherens junctions increases the availability of nutrients to tissues and facilitates leukocyte trafficking and diapedesis (10, 12, 17, 55). The importance of endothelial cell barrier integrity is often in conflict with requirements for endothelial cells to move in order to permit angiogenesis and repair or cell and fluid egress, and as a result, VEGF-induced VE-cadherin responses are tightly controlled (10, 17, 18, 32, 33, 59). This limits capillary permeability while dynamically responding to a variety of endothelial cell-specific factors and conditions. However, if unregulated, this process can result in localized capillary permeability and edema (2, 9, 10, 12, 14, 17, 29, 60).Interestingly, tissue edema and hypoxia are common findings in both HPS and HFRS patients (11, 31, 62), and the ability of pathogenic hantaviruses to infect human endothelial cells provides a means for hantaviruses to directly alter normal VEGF-VE-cadherin regulation. In fact, the permeability of endothelial cells infected by pathogenic Andes virus (ANDV) or Hantaan virus (HTNV) is dramatically enhanced in response to VEGF addition (22). This response is absent from endothelial cells comparably infected with the nonpathogenic TULV and suggests that enhanced VEGF-induced endothelial cell permeability is a common underlying response of both HPS- and HFRS-causing hantaviruses (22). In these studies, we comparatively investigate responses of human endothelial cells infected with pathogenic ANDV and HTNV, as well as nonpathogenic TULV.     

Tropomyosin Isoform Expression Regulates the Transition of Adhesions To Determine Cell Speed and Direction

The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.Among the different modes of migration that cells adopt, mesenchymal cell migration is dependent on integrin-based adhesion to the extracellular matrix (14), and the cellular mechanisms regulating integrin adhesion formation and turnover (adhesion dynamics) are integral to this process. The fate of integrin adhesions is intimately linked with filaments of polymerized actin (4). At the molecular level, actin filaments are highly dynamic, and this aspect of actin polymer biology provides an important control mechanism by which cells can organize filaments into structures with distinct properties. Tropomyosins are a multi-isoform family of actin-associating proteins that confer isoform-specific regulation of diverse actin filaments (3, 16, 34, 35). The interdependence of integrin adhesions and actin filaments suggests that expression of actin-associated proteins such as the tropomyosins may represent a mechanism for the regulation of adhesion dynamics that determine cell migration.In migrating cells small integrin-based focal complexes form at the periphery of lamellipodial extensions (32). These complexes are characterized by their subcellular distribution, dot-like shape, dependence on Rac activity, phosphorylated paxillin, and association with the network of short, branched actin filaments at the leading edge. The focal complexes are short lived (43) but provide strong traction forces at the leading edge (2) and most likely regulate directional migration (19). Subsets of focal complexes mature into focal adhesions, structures characterized by: Rho GTPase and Rho kinase dependence, dash-like shape, high levels of paxillin and phosphorylated paxillin, and low levels of the actin-binding molecule tensin (43, 44). The focal adhesions play an important role in anchoring bundles of polymerized actin stress fibers, providing the contractile force necessary for the translocation of the cell body during migration. There are at least three distinct classes of stress fibers observed in migrating cells (20, 27). Dorsal stress fibers are inserted into focal adhesions at the ventral surface of the cell. The distal end of the dorsal fibers can associate with a second type of actin fiber, the transverse arcs that run parallel to the leading edge and are not directly connected to focal adhesions. Ventral stress fibers have focal adhesions at either end and can be established following the contraction of two dorsal stress fibers and the associated transverse arc to form one actin bundle (20).Increased ventral stress fibers and focal adhesions are characteristic of nonmotile cells, in contrast, cell migration depends on focal adhesion turnover at the leading edge, allowing the formation of newly protruding regions of membrane and focal complex formation (28, 39). While the precise mechanism of focal adhesion turnover is incompletely understood, activation and phosphorylation of Src kinase, p130Cas, and paxillin (13, 39, 45) have all been implicated in focal adhesion turnover. A biphasic relationship between cell adhesion and cell speed suggests that conditions that alter the turnover rate of focal adhesions (either too much or too little) can reduce cell speed (18, 22).In cells with a fibroblastic phenotype, increased levels of acto-myosin contractility promote focal adhesion transition to fibrillar adhesions (also known as ECM contacts) (6, 7): elongated, thin, central arrays of dots or elongated fibrils that characteristically contain tensin but low levels of phosphorylated paxillin (29, 44, 45) and bind fibrils of fibronectin parallel to actin bundles (23, 29). These adhesions are formed by ligand-occupied fibronectin integrin receptor translocation from focal adhesions along bundles of actin filaments toward the cell center, and the process is dependent on an intact actin cytoskeleton and myosin activity (29). Receptor translocation stimulates matrix reorganization by transmitting cytoskeleton-generated tension through the integrin receptors onto the surrounding matrix (25, 29). The rate of receptor translocation is apparently independent from the rate of cell migration (29). However, the cytoskeletal tension that causes the fibrillar adhesion formation is also reported to decrease paxillin phosphorylation (45). Since phosphorylated paxillin is required for the generation of new focal complexes (45), conditions which switch the balance of adhesion in favor of fibrillar adhesion should presumably result in significantly reduced paxillin phosphorylation, leading to reduced focal adhesion turnover and correspondingly decreased cell migration.The cytoskeletal tropomyosin Tm5NM1 is a broadly distributed isoform (37) that alters cell shape (34), localizes to and promotes stress fibers that are resistant to actin depolymerizing drugs (9), enhances myosin IIA activation and recruitment to stress fibers, and inhibits cell migration (3). Therefore, we hypothesized that Tm5NM1 expression might determine cell migration by coordinating actin-dependent transition toward a predominance of focal adhesions and fibrillar adhesions. Using overexpression, knockdown, and genetic knockout models, we demonstrate that Tm5NM1 inhibits cell migration by promoting selective stabilization of focal adhesions and transition to fibrillar adhesions via the regulation of paxillin phosphorylation.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.     

Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP

Cell migration is critical for normal development and for pathological processes including cancer cell metastasis. Dynamic remodeling of focal adhesions and the actin cytoskeleton are crucial determinants of cell motility. The Rho family and the mitogen-activated protein kinase (MAPK) module consisting of MEK-extracellular signal-regulated kinase (ERK) are important regulators of these processes, but mechanisms for the integration of these signals during spreading and motility are incompletely understood. Here we show that ERK activity is required for fibronectin-stimulated Rho-GTP loading, Rho-kinase function, and the maturation of focal adhesions in spreading cells. We identify p190A RhoGAP as a major target for ERK signaling in adhesion assembly and identify roles for ERK phosphorylation of the C terminus in p190A localization and activity. These observations reveal a novel role for ERK signaling in adhesion assembly in addition to its established role in adhesion disassembly.Cell migration is a highly coordinated process essential for physiological and pathological processes (69). Signaling through Rho family GTPases (e.g., Rac, Cdc42, and Rho) is crucial for cell migration. Activated Rac and Cdc42 are involved in the production of a dominant lamellipodium and filopodia, respectively, whereas Rho-stimulated contractile forces are required for tail retraction and to maintain adhesion to the matrix (57, 58, 68). Rac- and Cdc42-dependent membrane protrusions are driven by the actin cytoskeleton and the formation of peripheral focal complexes; Rho activation stabilizes protrusions by stimulating the formation of mature focal adhesions and stress fibers. Active Rho influences cytoskeletal dynamics through effectors including the Rho kinases (ROCKs) (2, 3).Rho activity is stimulated by GEFs that promote GTP binding and attenuated by GTPase-activating proteins (GAPs) that enhance Rho''s intrinsic GTPase activity. However, due to the large number of RhoGEFs and RhoGAPs expressed in mammalian cells, the molecular mechanisms responsible for regulation of Rho activity in time and space are incompletely understood. p190A RhoGAP (hereafter p190A) is implicated in adhesion and migration signaling. p190A contains an N-terminal GTPase domain, a large middle domain juxtaposed to the C-terminal GAP domain, and a short C-terminal tail (74). The C-terminal tail of ∼50 amino acids is divergent between p190A and the closely related family member p190B (14) and thus may specify the unique functional roles for p190A and p190B revealed in gene knockout studies (10, 11, 41, 77, 78). p190A activity is dynamically regulated in response to external cues during cell adhesion and migration (5, 6, 59). Arthur et al. (5) reported that p190A activity is required for the transient decrease in RhoGTP levels seen in fibroblasts adhering to fibronectin. p190A activity is positively regulated by tyrosine phosphorylation (4, 5, 8, 17, 31, 39, 40, 42): phosphorylation at Y1105 promotes its association with p120RasGAP and subsequent recruitment to membranes or cytoskeleton (8, 17, 27, 31, 71, 75, 84). However, Y1105 phosphorylation is alone insufficient to activate p190A GAP activity (39). While the functions of p190A can be irreversibly terminated by ubiquitinylation in a cell-cycle-dependent manner (80), less is known about reversible mechanisms that negatively regulate p190A GAP activity during adhesion and motility.The integration of Rho family GTPase and extracellular signal-regulated kinase (ERK) signaling is important for cell motility (48, 50, 63, 76, 79). Several studies have demonstrated a requirement for ERK signaling in the disassembly of focal adhesions in migrating cells, in part through the activation of calpain proteases (36, 37) that can downregulate focal adhesion kinase (FAK) signaling (15), locally suppress Rho activity (52), and sever cytoskeletal linkers to focal adhesions (7, 33). Inhibition of ERK signaling increases focal adhesion size and retards disassembly of focal adhesions in adherent cells (57, 64, 85, 86). It is also recognized that ERK modulates Rho-dependent cellular processes, including membrane protrusion and migration (18, 25, 64, 86). Interestingly, ERK activated in response to acute fibronectin stimulation localizes not only to mature focal adhesions, but also to peripheral focal complexes (32, 76). Since these complexes can either mature or be turned over (12), ERK may play a distinct role in focal adhesion assembly. ERK is proposed to promote focal adhesion formation by activating myosin light chain kinase (MLCK) (21, 32, 50).Here we find that ERK activity is required for Rho activation and focal adhesion formation during adhesion to fibronectin and that p190A is an essential target of ERK signaling in this context. Inspection of the p190A C terminus reveals a number of consensus ERK sites and indeed p190A is phosphorylated by recombinant ERK only on its C terminus in vitro, and on the same C-terminal peptide in vivo. Mutation of the C-terminal ERK phosphorylation sites to alanine increases the biochemical and biological activity of p190A. Finally, inhibition of MEK or mutation of the C-terminal phosphorylation sites enhances retention of p190A in peripheral membranes during spreading on fibronectin. Our data support the conclusion that ERK phosphorylation inhibits p190A allowing increases in RhoGTP and cytoskeletal changes necessary for focal adhesion formation.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

HIV-1 Nef Inhibits Ruffles,Induces Filopodia,and Modulates Migration of Infected Lymphocytes

The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal “axial tomography,” this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.Human immunodeficiency virus type 1 (HIV-1) mostly replicates in T-cell areas of secondary lymphoid organs (SLOs) and induces pathological changes in their architecture. Such changes are likely due to a combination of events, including destruction of T cells, chronic immune activation, and alteration of T-cell motility toward and inside the SLOs (27, 37, 50, 53). Indeed, to fulfill their immune surveillance role, T cells continuously circulate in and out of blood, lymph nodes (LNs), and tissues (60).Lymphocyte recruitment from the bloodstream into LNs depends on three distinct processes, i.e., attachment to high endothelial venules (HEVs), extravasation, and cell migration (10, 60). Adhesion to the endothelium and extracellular matrix (ECM) is a crucial step, regulated in part by β1 integrins, α4β1 (VLA-4) and α5β1, that bind VCAM-1 and/or fibronectin (56). Chemokines and their Gαi-protein-coupled receptors are key regulators of lymphocyte trafficking (32). For instance, CCL19 and CCL21 are constitutively produced by HEVs and by fibroblastic reticular cells of T-cell areas of LNs (21, 28, 29). These two chemokines share the receptor CCR7, expressed by naïve T cells and a fraction of memory T cells (47). They play a major role in lymphocyte homing to LNs, in steady state as well as under conditions of inflammation, and may control T-cell positioning within defined functional compartments (1, 17, 18, 47). CXCR4 and its ligand CXCL12/SDF-1 also contribute to T-cell entry into LNs (5, 23, 40). In addition, effector and memory T cells express a broad range of receptors binding inflammatory chemokines, such as the CCR5 ligands CCL3 (MIP1α), CCL4 (MIP1β), and CCL5 (Rantes).Efficient accomplishment of lymphocyte migration and immune functions requires tight regulation of the cellular cytoskeleton (59). This is mediated by the small GTPases of the Rho subfamily, such as Rho, Rac, and Cdc42 (11, 58). They activate specific actin filament assembly factors to generate sheet-like protrusive structures (such as lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli) (6). These structures have different functions. Lamellipodia and ruffles are formed during crawling cell motility and spreading. Filopodia protrude from the leading edges of many motile cells. They appear to perform sensory and exploratory functions to steer cells, depending on cues from the environment (42). Moreover, filopodia, or other thin structures called tunneling nanotubes, have been shown to form intercellular bridges, allowing viruses to spread through remote contacts between infected cells and targets (44, 48, 49, 52).HIV-1 hijacks cytoskeleton dynamics in order to ensure viral entry and transport within and egress from target cells (34; reviewed in reference 13). In particular, the viral protein Nef modifies actin remodeling in various cell systems. In T cells, Nef alters actin rearrangements triggered by activation of T-cell (TCR) or chemokine receptors (22, 54). Nef inhibits immunological synapse formation, a dynamic process involving rapid actin modifications (57). Nef also affects plasma membrane plasticity, inducing secretion of microvesicle clusters (33). In macrophages, Nef induces the extension of long intercellular conduits allowing its own transfer to B cells (61). A number of studies have reported that Nef affects T-cell chemotaxis (generally to CXCL12) through the modulation of Rho-GTPase-regulated signaling pathways (7, 24, 39, 54). Migration studies have generally been performed using Nef-expressing cells, and rarely in the context of HIV-1 infection (54). From a molecular standpoint, it has recently been proposed that Nef acts in part by deregulating cofilin, an actin-depolymerizing factor that promotes actin turnover and subsequent cell motility (54).In the present study, our goal was to gain further insights into the effect of HIV-1 infection on cytoskeleton dynamics. We used a panel of innovative techniques allowing analysis of cell shape, adhesion, and motility. We report that in HIV-infected lymphocytes, Nef promotes filopodium-like formation while it inhibits membrane ruffling. Nef impairs cell adhesion on the extracellular matrix and decreases intrinsic cell motility. Lymphocyte migration toward various chemokines (CXCL12, CCL3, and CCL19) is also inhibited. Our results suggest that Nef may facilitate viral spread and contribute to AIDS pathogenesis by manipulating the migration of lymphocytes.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Organization of F-Actin via Concerted Regulation of Kette by PTP61F and dAbl

We identify Kette, a key regulator of actin polymerization, as a substrate for Drosophila protein tyrosine phosphatase PTP61F, as well as for dAbl tyrosine kinase. We further show that dAbl is a direct substrate for PTP61F. Therefore, Kette phosphotyrosine levels are regulated both directly and indirectly by PTP61F. Kette and PTP61F genetically interact in the regulation of F-actin organization in pupal eye discs, suggesting that tyrosine phosphorylation is essential for the proper regulation of Kette-mediated actin dynamics. This hypothesis was confirmed by demonstrating the loss of Kette-mediated F-actin organization and lamella formation in S2 cells in a Kette Y482F mutant in which the dAbl phosphorylation site was eliminated. Our results establish for the first time that PTP61F and dAbl ensure proper actin organization through the coordinated and reversible tyrosine phosphorylation of Kette.The actin cytoskeleton is regulated as a function of development, cell motility, intracellular transport, and the cell cycle by the polymerization of G-actin to F-actin (34). Correct regulation of actin cytoskeletal dynamics is essential to numerous differentiating and cellular processes in the nervous system (9) and musculature (42), among others. Actin polymerization is regulated by a number of proteins, among which is human NCK-associated protein 1 (NAP-1 [3, 4, 45]). It and its Drosophila orthologue, Kette (Hem in FlyBase), are critical components in both SCAR/WAVE and WASP complexes, which play essential roles in transducing Rac1 signals to initiate Arp2/3-dependent actin polymerization (6, 25, 40, 48). Murine NAP-1 interacts with NCK, an SH2-SH3 adaptor protein (4), and is essential for proper neuronal differentiation in the cortex (53). Neuronal differentiation and neural tube defects are observed in NAP-1 mutant mice, apparently due to reduced localization of WAVE1 to the cell membrane (53).In Drosophila, loss of kette activity specifically results in the accumulation of cytosolic F-actin (6). Kette protein associates with F-actin in the cytosol, but also at focal contact sites, where it apparently antagonizes SCAR/WAVE function and activates WASP-dependent actin polymerization (6). Despite its role in repressing SCAR/WAVE function, Kette serves to protect the complex from proteosome-mediated degradation and is critical to its intracellular localization (25). At the level of the organism, kette alleles affect axonal growth and pathfinding due to aberrant actin cytoskeleton formation, for example, altering crossing of the embryonic ventral midline by VUM neuron axons, as well as generating aberrant axonal projections in both motor and sensory neurons (21). Like mammalian NAP-1, Drosophila kette also interacts with the fly NCK orthologue, dreadlocks (dock) (21). Other evidence for the conserved interaction of Kette with signaling cascades is provided by the observation that kette mutant phenotypes are partially rescuable by overexpression of the small G protein Rac1 (21). The interaction of kette with dock suggests the possibility of tyrosine phosphorylation in the regulation of Kette activity, but no evidence supporting this hypothesis has been reported.Signaling by tyrosine phosphorylation in various metazoans controls numerous processes involved in cellular differentiation and proliferation. Many of the components regulating tyrosine phosphorylation have been identified and characterized using genetic, biochemical, molecular, and genomic sequence analyses (31). However, in contrast to the very well-characterized regulation of cellular processes by kinase-mediated tyrosine phosphorylation (15, 52), their regulation by dephosphorylation by protein tyrosine phosphatases (PTPs) has generally lagged behind. Although the functions of several receptor PTPs have been clearly defined as playing essential roles in axon guidance in both Drosophila (12, 23, 41, 47, 50) and mammals (44, 49), our understanding of nontransmembrane PTPs (NT-PTPs) is more limited. Only three of the eight putative Drosophila NT-PTPs have been characterized genetically. Corkscrew (Csw) acts as a downstream effector of various receptor protein tyrosine kinases (PTKs) and is essential for R7 photoreceptor development (35). PTP-enhancer of Ras1 has been characterized as an essential regulator antagonizing signaling mediated by Ras1, possibly through tyrosine dephosphorylation of mitogen-activated protein kinase (24, 36). More recently, it has been shown that PTP-meg participates in the establishment and maintenance of axon projections in the Drosophila brain (51). Other than these, the functions of Drosophila NT-PTPs remain largely unknown.PTP61F was originally identified as an NT-PTP that contains one phosphatase domain in the N-terminal region and five proline-rich motifs in the C-terminal tail (29). It is the Drosophila orthologue of mammalian PTP1B and T-cell PTP (TC-PTP) (1), which have been implicated in the regulation of signaling by both insulin (39) and JAK/STAT (33). Two PTP61F isoforms due to alternative splicing possess unique sequences at the C terminus, which determine either internal membrane-association (PTP61Fm) or nuclear localization (PTP61Fn) (29). To date, limited data suggest that PTP61F may participate in the downregulation of JAK/STAT signaling (2, 32), although the underlying mechanism remains unexplored. While PTP61F may recognize the adaptor proteins DOCK (10) and Abi (20) as potential substrates, the signaling pathways involving these interactions have not been clearly defined. In this study, we demonstrate for the first time that the regulation of Kette, and hence the localization and polymerization of the actin cytoskeleton, is achieved by reversible tyrosine phosphorylation under the control of both PTP61F and the PTK dAbl.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

HIV-1 possesses an exquisite ability to infect cells independently from their cycling status by undergoing an active phase of nuclear import through the nuclear pore. This property has been ascribed to the presence of karyophilic elements present in viral nucleoprotein complexes, such as the matrix protein (MA); Vpr; the integrase (IN); and a cis-acting structure present in the newly synthesized DNA, the DNA flap. However, their role in nuclear import remains controversial at best. In the present study, we carried out a comprehensive analysis of the role of these elements in nuclear import in a comparison between several primary cell types, including stimulated lymphocytes, macrophages, and dendritic cells. We show that despite the fact that none of these elements is absolutely required for nuclear import, disruption of the central polypurine tract-central termination sequence (cPPT-CTS) clearly affects the kinetics of viral DNA entry into the nucleus. This effect is independent of the cell cycle status of the target cells and is observed in cycling as well as in nondividing primary cells, suggesting that nuclear import of viral DNA may occur similarly under both conditions. Nonetheless, this study indicates that other components are utilized along with the cPPT-CTS for an efficient entry of viral DNA into the nucleus.Lentiviruses display an exquisite ability to infect dividing and nondividing cells alike that is unequalled among Retroviridae. This property is thought to be due to the particular behavior or composition of the viral nucleoprotein complexes (NPCs) that are liberated into the cytoplasm of target cells upon virus-to-cell membrane fusion and that allow lentiviruses to traverse an intact nuclear membrane (17, 28, 29, 39, 52, 55, 67, 79). In the case of the human immunodeficiency type I virus (HIV-1), several studies over the years identified viral components of such structures with intrinsic karyophilic properties and thus perfect candidates for mediation of the passage of viral DNA (vDNA) through the nuclear pore: the matrix protein (MA); Vpr; the integrase (IN); and a three-stranded DNA flap, a structure present in neo-synthesized viral DNA, specified by the central polypurine tract-central termination sequence (cPPT-CTS). It is clear that these elements may mediate nuclear import directly or via the recruitment of the host''s proteins, and indeed, several cellular proteins have been found to influence HIV-1 infection during nuclear import, like the karyopherin α2 Rch1 (38); importin 7 (3, 30, 93); the transportin SR-2 (13, 20); or the nucleoporins Nup98 (27), Nup358/RANBP2, and Nup153 (13, 56).More recently, the capsid protein (CA), the main structural component of viral nucleoprotein complexes at least upon their cytoplasmic entry, has also been suggested to be involved in nuclear import or in postnuclear entry steps (14, 25, 74, 90, 92). Whether this is due to a role for CA in the shaping of viral nucleoprotein complexes or to a direct interaction between CA and proteins involved in nuclear import remains at present unknown.Despite a large number of reports, no single viral or cellular element has been described as absolutely necessary or sufficient to mediate lentiviral nuclear import, and important controversies as to the experimental evidences linking these elements to this step exist. For example, MA was among the first viral protein of HIV-1 described to be involved in nuclear import, and 2 transferable nuclear localization signals (NLSs) have been described to occur at its N and C termini (40). However, despite the fact that early studies indicated that the mutation of these NLSs perturbed HIV-1 nuclear import and infection specifically in nondividing cells, such as macrophages (86), these findings failed to be confirmed in more-recent studies (23, 33, 34, 57, 65, 75).Similarly, Vpr has been implicated by several studies of the nuclear import of HIV-1 DNA (1, 10, 21, 43, 45, 47, 64, 69, 72, 73, 85). Vpr does not possess classical NLSs, yet it displays a transferable nucleophilic activity when fused to heterologous proteins (49-51, 53, 77, 81) and has been shown to line onto the nuclear envelope (32, 36, 47, 51, 58), where it can truly facilitate the passage of the viral genome into the nucleus. However, the role of Vpr in this step remains controversial, as in some instances Vpr is not even required for viral replication in nondividing cells (1, 59).Conflicting results concerning the role of IN during HIV-1 nuclear import also exist. Indeed, several transferable NLSs have been described to occur in the catalytic core and the C-terminal DNA binding domains of IN, but for some of these, initial reports of nuclear entry defects (2, 9, 22, 46, 71) were later shown to result from defects at steps other than nuclear import (60, 62, 70, 83). These reports do not exclude a role for the remaining NLSs in IN during nuclear import, and they do not exclude the possibility that IN may mediate this step by associating with components of the cellular nuclear import machinery, such as importin alpha and beta (41), importin 7 (3, 30, 93, 98), and, more recently, transportin-SR2 (20).The central DNA flap, a structure present in lentiviruses and in at least 1 yeast retroelement (44), but not in other orthoretroviruses, has also been involved in the nuclear import of viral DNA (4, 6, 7, 31, 78, 84, 95, 96), and more recently, it has been proposed to provide a signal for viral nucleoprotein complexes uncoating in the proximity of the nuclear pore, with the consequence of providing a signal for import (8). However, various studies showed an absence or weakness of nuclear entry defects in viruses devoid of the DNA flap (24, 26, 44, 61).Overall, the importance of viral factors in HIV-1 nuclear import is still unclear. The discrepancies concerning the role of MA, IN, Vpr, and cPPT-CTS in HIV-1 nuclear import could in part be explained by their possible redundancy. To date, only one comprehensive study analyzed the role of these four viral potentially karyophilic elements together (91). This study showed that an HIV-1 chimera where these elements were either deleted or replaced by their murine leukemia virus (MLV) counterparts was, in spite of an important infectivity defect, still able to infect cycling and cell cycle-arrested cell lines to similar efficiencies. If this result indicated that the examined viral elements of HIV-1 were dispensable for the cell cycle independence of HIV, as infections proceeded equally in cycling and arrested cells, they did not prove that they were not required in nuclear import, because chimeras displayed a severe infectivity defect that precluded their comparison with the wild type (WT).Nuclear import and cell cycle independence may not be as simply linked as previously thought. On the one hand, there has been no formal demonstration that the passage through the nuclear pore, and thus nuclear import, is restricted to nondividing cells, and for what we know, this passage may be an obligatory step in HIV infection in all cells, irrespective of their cycling status. In support of this possibility, certain mutations in viral elements of HIV affect nuclear import in dividing as well as in nondividing cells (4, 6, 7, 31, 84, 95). On the other hand, cell cycle-independent infection may be a complex phenomenon that is made possible not only by the ability of viral DNA to traverse the nuclear membrane but also by its ability to cope with pre- and postnuclear entry events, as suggested by the phenotypes of certain CA mutants (74, 92).Given that the cellular environment plays an important role during the early steps of viral infection, we chose to analyze the role of the four karyophilic viral elements of HIV-1 during infection either alone or combined in a wide comparison between cells highly susceptible to infection and more-restrictive primary cell targets of HIV-1 in vivo, such as primary blood lymphocytes (PBLs), monocyte-derived macrophages (MDM), and dendritic cells (DCs).In this study, we show that an HIV-1-derived virus in which the 2 NLSs of MA are mutated and the IN, Vpr, and cPPT-CTS elements are removed displays no detectable nuclear import defect in HeLa cells independently of their cycling status. However, this mutant virus is partially impaired for nuclear entry in primary cells and more specifically in DCs and PBLs. We found that this partial defect is specified by the cPPT-CTS, while the 3 remaining elements seem to play no role in nuclear import. Thus, our study indicates that the central DNA flap specifies the most important role among the viral elements involved thus far in nuclear import. However, it also clearly indicates that the role played by the central DNA flap is not absolute and that its importance varies depending on the cell type, independently from the dividing status of the cell.