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.     

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).     

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.     

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.     

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.     

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.     

Hollow-Fiber Membrane Chamber as a Device for In Situ Environmental Cultivation

A hollow-fiber membrane chamber (HFMC) was developed as an in situ cultivation device for environmental microorganisms. The HFMC system consists of 48 to 96 pieces of porous hollow-fiber membrane connected with injectors. The system allows rapid exchange of chemical compounds, thereby simulating a natural environment. Comparative analysis through the cultivation of three types of environmental samples was performed using this newly designed device and a conventional agar-based petri dish. The results show that the ratios of novel phylotypes in isolates, species-level diversities, and cultivabilities in HFMC-based cultivation are higher than those in an agar-based petri dish for all three samples, suggesting that the new in situ cultivation device is effective for cultivation of various environmental microorganisms.Although highly diverse untapped microbial consortia exist in natural environments, it is generally recognized that most microorganisms are not readily cultivable in the laboratory (1, 17). Recent advances in culture-independent molecular approaches, based on rRNA or genomic approaches that can estimate microbial composition and function, have considerably improved knowledge of microbial ecosystems (7, 11, 29, 32). However, cultivation-based approaches are still necessary for comprehensive elucidation of the physiology and ecology of these organisms and for their biotechnological applications. Recently, several attempts have been made to address these issues (19, 24). Modification of growth conditions based on conventional methods, such as controlling the substrate composition and concentration, the gelling reagent, trace additives such as signaling molecules, and the length of cultivation, has improved isolation efficiencies of rarely cultivated phyla and increased the diversity of isolates (3, 4, 6, 9, 14, 15, 26, 28, 30). Newly developed cultivation methods such as high-throughput methods have brought success with uncultivated microorganisms and improved cultivation capabilities (5, 8, 20, 22, 35). Additionally, development and use of a diffusion chamber to enable the exchange of chemical compounds during cultivation have demonstrated the importance of in situ environmental conditions for the isolation of environmental microorganisms (2, 16). Among them, a concept based on “environmental simulation” is likely to be generally effective for cultivation of environmental microorganisms because various factors that are unknown but necessary for recovery and growth can be provided to the microorganisms (10). However, very few methods have been developed that are applicable to cultivation of microorganisms under in situ environmental conditions. Consequently, it is still important to develop a new cultivation device that is particularly suitable for pure cultivation under in situ environmental conditions while maintaining simple operation. For this study, we designed a new cultivation device, called the hollow-fiber membrane chamber (HFMC), which can provide in situ environmental and liquid culture conditions while maintaining a microliter- to milliliter-scale volume of each chamber. We evaluated the effect of the new device, especially for cultivation under in situ environmental conditions, on cultivation of samples from several different environments.     

Ebola Virus Glycoprotein Counteracts BST-2/Tetherin Restriction in a Sequence-Independent Manner That Does Not Require Tetherin Surface Removal

BST-2/tetherin is an interferon-inducible protein that restricts the release of enveloped viruses from the surface of infected cells by physically linking viral and cellular membranes. It is present at both the cell surface and in a perinuclear region, and viral anti-tetherin factors including HIV-1 Vpu and HIV-2 Env have been shown to decrease the cell surface population. To map the domains of human tetherin necessary for both virus restriction and sensitivity to viral anti-tetherin factors, we constructed a series of tetherin derivatives and assayed their activity. We found that the cytoplasmic tail (CT) and transmembrane (TM) domains of tetherin alone produced its characteristic cellular distribution, while the ectodomain of the protein, which includes a glycosylphosphatidylinositol (GPI) anchor, was sufficient to restrict virus release when presented by the CT/TM regions of a different type II membrane protein. To counteract tetherin restriction and remove it from the cell surface, HIV-1 Vpu required the specific sequence present in the TM domain of human tetherin. In contrast, the HIV-2 Env required only the ectodomain of the protein and was sensitive to a point mutation in this region. Strikingly, the anti-tetherin factor, Ebola virus GP, was able to overcome restriction conferred by both tetherin and a series of functional tetherin derivatives, including a wholly artificial tetherin molecule. Moreover, GP overcame restriction without significantly removing tetherin from the cell surface. These findings suggest that Ebola virus GP uses a novel mechanism to circumvent tetherin restriction.Pathogenic viruses often have evolved mechanisms to neutralize host defenses that act at the cellular level to interfere with the virus life cycle. Such cellular restriction factors have been most extensively characterized for HIV-1 (38) and include the interferon-inducible membrane protein BST-2/HM1.24/CD317/tetherin (28, 40). If unchecked, tetherin blocks the release of newly formed HIV-1 particles from cells by physically tethering them at the cell surface (7, 28, 32, 40). In addition, tetherin has been shown to act against a broad range of enveloped viral particles, including retroviruses, filoviruses, arenaviruses, and herpesviruses (17, 18, 23, 35). In turn, certain viruses that are targeted by tetherin appear to have evolved counteracting activities, and anti-tetherin factors so far identified include HIV-1 Vpu; HIV-2 Env; simian immunodeficiency virus (SIV) Nef, Vpu, and Env proteins; Ebola virus GP; and Kaposi''s sarcoma-associated herpesvirus (KSHV) K5 (11, 16, 18, 20, 23, 28, 36, 40, 44, 45).Tetherin is a homodimeric type II integral membrane protein containing an N-terminal cytoplasmic tail (CT), a single-pass transmembrane domain (TM), an ectodomain-containing predicted coiled-coil regions, two glycoslyation sites, three conserved cysteines, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (2, 19, 31). This unusual topology, with two independent membrane anchors, has led to the suggestion that the retention of virions at the cell surface arises from tetherin''s ability to be inserted simultaneously in both host and viral membranes (28, 32, 41) or, alternatively, that dimers or higher-order complexes of tetherin conferred by the ectodomain mediate this effect (39). Interestingly, an artificial tetherin containing the same structural features as the native protein but constructed from unrelated sequences was able to restrict both HIV-1 and Ebola virus particles (32). This suggests that the viral lipid envelope is the target of tetherin and provides an explanation for tetherin''s broad activity against diverse enveloped viruses.A fraction of tetherin is present at the plasma membrane of cells (9, 14), and it has been proposed that viral anti-tetherin factors function by removing this cell surface fraction (40). This now has been shown to occur in the presence of HIV-1 Vpu (5, 7, 15, 26, 34, 40, 44), HIV-2 Env (5, 20), SIV Env (11), SIV Nef (15), and KSHV K5 (3, 23). In addition, certain anti-tetherin factors also may promote the degradation of tetherin, as has been observed for both HIV-1 Vpu (3, 5, 7, 10, 22, 26, 27) and KSHV K5 (3, 23), although Vpu also appears able to block tetherin restriction in the absence of degradation (8), and no effects on tetherin steady-state levels have been observed in the presence of either the HIV-2 or SIVtan Env (11, 20). Simply keeping tetherin away from the cell surface, or targeting it for degradation, may not be the only mechanism used by anti-tetherin factors, since it also has been reported that Vpu does not affect the levels of surface tetherin or its total cellular levels in certain T-cell lines (27).The interactions between tetherin and viral anti-tetherin factors show evidence of species specificity, suggesting ongoing evolution between viruses and their hosts. HIV-1 Vpu is active against human and chimpanzee tetherin but not other primate tetherins (10, 25, 34, 36, 44, 45), while SIV Nef proteins are active against primate but not human tetherins (16, 36, 44, 45). This suggests that, unlike tetherin restriction, the action of the anti-tetherin factors may involve specific sequence interactions. Indeed, the TM domain has been recognized as a target for HIV-1 Vpu (10, 15, 16, 25, 34), while a single point mutation introduced into the extracellular domain of human tetherin can block its antagonism by the SIVtan Env (11).In the present study, we investigated the roles of the different domains of tetherin in both promoting virus restriction and conferring susceptibility to the anti-tetherin factors encoded by HIV-1, HIV-2, and Ebola virus. We confirmed that tetherin restriction can be conferred by proteins that retain the two distinct membrane anchors, while signals for the cellular localization of the protein reside in the CT/TM domains of the protein. We found that the Vpu protein targets the TM domain of tetherin, while the HIV-2 Env targets the ectodomain of the protein. In contrast, the Ebola virus GP appears to use a non-sequence-specific mechanism to counteract tetherin restriction, since even an artificial tetherin could be successfully overcome by GP expression. Interestingly, Ebola virus GP counteracted tetherin restriction without removing the protein from the cell surface, suggesting that it is possible to overcome this restriction by mechanisms other than blocking tetherin''s cell surface expression.     

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.