Structural and biochemical basis of interdependent FANCI-FANCD2 ubiquitination.

Di-monoubiquitination of the FANCI-FANCD2 (ID2) complex is a central and crucial step for the repair of DNA interstrand crosslinks via the Fanconi anaemia pathway. While FANCD2 ubiquitination precedes FANCI ubiquitination, FANCD2 is also deubiquitinated at a faster rate than FANCI, which can result in a FANCI-ubiquitinated ID2 complex (IUb D2). Here, we present a 4.1 Å cryo-EM structure of IUb D2 complex bound to double-stranded DNA. We show that this complex, like ID2Ub and IUb D2Ub , is also in the closed ID2 conformation and clamps on DNA. The target lysine of FANCD2 (K561) becomes fully exposed in the IUb D2-DNA structure and is thus primed for ubiquitination. Similarly, FANCI's target lysine (K523) is also primed for ubiquitination in the ID2Ub -DNA complex. The IUb D2-DNA complex exhibits deubiquitination resistance, conferred by the presence of DNA and FANCD2. ID2Ub -DNA, on the other hand, can be efficiently deubiquitinated by USP1-UAF1, unless further ubiquitination on FANCI occurs. Therefore, FANCI ubiquitination effectively maintains FANCD2 ubiquitination in two ways: it prevents excessive FANCD2 deubiquitination within an IUb D2Ub -DNA complex, and it enables re-ubiquitination of FANCD2 within a transient, closed-on-DNA, IUb D2 complex.

Coinfection by influenza A virus and respiratory syncytial virus produces hybrid virus particles.

Interactions between respiratory viruses during infection affect transmission dynamics and clinical outcomes. To identify and characterize virus-virus interactions at the cellular level, we coinfected human lung cells with influenza A virus (IAV) and respiratory syncytial virus (RSV). Super-resolution microscopy, live-cell imaging, scanning electron microscopy and cryo-electron tomography revealed extracellular and membrane-associated filamentous structures consistent with hybrid viral particles (HVPs). We found that HVPs harbour surface glycoproteins and ribonucleoproteins of IAV and RSV. HVPs use the RSV fusion glycoprotein to evade anti-IAV neutralizing antibodies and infect and spread among cells lacking IAV receptors. Finally, we show that IAV and RSV coinfection in primary cells of the bronchial epithelium results in viral proteins from both viruses co-localizing at the apical cell surface. Our observations define a previously unknown interaction between respiratory viruses that might affect virus pathogenesis by expanding virus tropism and enabling immune evasion.

Stimuli-Responsive Aggregation of High Molar Mass Poly(N,N-Diethylacrylamide)-b-Poly(4-Acryloylmorpholine) in Tetrahydrofuran.

The self-assembly of block copolymers constitutes a timely research area in polymer science with implications for applications like sensing or drug-delivery. Here, the unprecedented aggregation behavior of high molar mass block copolymer poly(N,N-diethylacrylamide)-b-poly(4-acryloylmorpholine) (PDEA-b-PAM) (Mn >400 kg mol-1 ) in organic solvent tetrahydrofuran (THF) is investigated. To elucidate the aggregation, dynamic light scattering, cryo-transmission electron microscopy, and turbidimetry are employed. The aggregate formation is assigned to the unprecedented upper critical solution temperature behavior of PAM in THF at elevated concentrations (> 6 wt.%) and high molar masses. Various future directions for this new thermo-responsive block copolymer are envisioned, for example, in the areas of photonics or templating of inorganic structures.

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers.

Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles.

Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA.

The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double-stranded DNA via promoting a large-scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary "Arm" ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.

TGFß inhibition restores a regenerative response in acute liver injury by suppressing paracrine senescence.

Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy. However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction of paracrine senescence was observed within 24 hours and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence, we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended on macrophage-derived transforming growth factor-ß1 (TGFß1) ligand. In acetaminophen poisoning, inhibition of TGFß receptor 1 (TGFßR1) improved mouse survival. TGFßR1 inhibition reduced senescence and enhanced liver regeneration even when delivered beyond the therapeutic window for treating acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

Structure and assembly of the Ebola virus nucleocapsid.

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical nucleocapsid. The nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of nucleocapsid or nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus nucleocapsid within intact viruses and recombinant nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.

CXCR2 Inhibition Profoundly Suppresses Metastases and Augments Immunotherapy in Pancreatic Ductal Adenocarcinoma.

CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.

Atypical chemokine receptor ACKR2 mediates chemokine scavenging by primary human trophoblasts and can regulate fetal growth, placental structure, and neonatal mortality in mice.

Inflammatory chemokines produced in the placenta can direct the migration of placental leukocytes using chemokine receptors that decorate the surface of these cells. Fetal trophoblasts can also express receptors for inflammatory chemokines, and they are one of the few cell types that express atypical chemokine receptor 2 (ACKR2), previously known as D6. ACKR2 binds many inflammatory CC chemokines but cannot stimulate cell migration or activate signaling pathways used by conventional chemokine receptors. Existing evidence suggests that ACKR2 is a specialized chemokine scavenger, but its function in primary human trophoblasts has not been explored. In mice, ACKR2 is thought to be dispensable for the reproductive success of unchallenged females that have conceived naturally, but it can suppress inflammation-induced abortion and aid the survival of implanted allogeneic embryos. In this article, we demonstrate that cultured primary human trophoblasts express ACKR2 far more strongly than genes encoding conventional receptors for inflammatory CC chemokines. Moreover, these cells are capable of the rapid internalization and efficient scavenging of extracellular chemokine, and this is mediated by ACKR2. We also report that in unchallenged DBA/1j mice, Ackr2 deficiency increases the incidence of stillbirth and neonatal death, leads to structural defects in the placenta, and can decrease fetal weight. Loss of Ackr2 specifically from fetal cells makes a key contribution to the placental defects. Thus, primary human trophoblasts use ACKR2 to scavenge chemokines, and ACKR2 deficiency can cause abnormal placental structure and reduced neonatal survival.

Inhibition of CXCR2 profoundly suppresses inflammation-driven and spontaneous tumorigenesis.

The chemokine receptor CXCR2 is a key mediator of neutrophil migration that also plays a role in tumor development. However, CXCR2 influences tumors through multiple mechanisms and might promote or inhibit tumor development depending on context. Here, we used several mouse models of spontaneous and inflammation-driven neoplasia to define indispensable roles for CXCR2 in benign and malignant tumors. CXCR2-activating chemokines were part of the secretome of cultured primary benign intestinal adenomas (ApcMin/+) and highly expressed by all tumors in all models. CXCR2 deficiency profoundly suppressed inflammation-driven tumorigenesis in skin and intestine as well as spontaneous adenocarcinoma formation in a model of invasive intestinal adenocarcinoma (AhCreER;Apcfl/+;Ptenfl/fl mice). Pepducin-mediated CXCR2 inhibition reduced tumorigenesis in ApcMin/+ mice. Ly6G+ neutrophils were the dominant source of CXCR2 in blood, and CXCR2 deficiency attenuated neutrophil recruitment. Moreover, systemic Ly6G+ cell depletion purged CXCR2-dependent tumor-associated leukocytes, suppressed established skin tumor growth and colitis-associated tumorigenesis, and reduced ApcMin/+ adenoma formation. CXCR2 is thus a potent protumorigenic chemokine receptor that directs recruitment of tumor-promoting leukocytes into tissues during tumor-inducing and tumor-driven inflammation. Similar leukocyte populations were also found in human intestinal adenomas, which suggests that CXCR2 antagonists may have therapeutic and prophylactic potential in the treatment of cancer.

The atypical chemokine receptor D6 contributes to the development of experimental colitis.

Proinflammatory CC chemokines control leukocyte recruitment and function during inflammation by engaging chemokine receptors expressed on circulating leukocytes. The D6 chemokine receptor can bind several of these chemokines, but appears unable to couple to signal transduction pathways or direct cell migration. Instead, D6 has been proposed to act as a chemokine scavenger, removing proinflammatory chemokines to dampen leukocyte responses. In this study, we have examined the role of D6 in the colon using the dextran sodium sulfate-induced model of colitis. We show that D6 is expressed in the resting colon, predominantly by stromal cells and B cells, and is up-regulated during colitis. Unexpectedly, D6-deficient mice showed reduced susceptibility to colitis and had less pronounced clinical symptoms associated with this model. D6 deletion had no impact on the level of proinflammatory CC chemokines released from cultured colon explants, or on the balance of leukocyte subsets recruited to the inflamed colon. However, late in colitis, inflamed D6-deficient colons showed enhanced production of several proinflammatory cytokines, including IFN-gamma and IL-17A, and there was a marked increase in IL-17A-secreting gammadelta T cells in the lamina propria. Moreover, Ab-mediated neutralization of IL-17A worsened the clinical symptoms of colitis at these later stages of the response in D6-deficient, but not wild-type, mice. Thus, D6 can contribute to the development of colitis by regulating IL-17A secretion by gammadelta T cells in the inflamed colon.

ACRP30 is secreted from 3T3-L1 adipocytes via a Rab11-dependent pathway.

Adipocytes are now known to secrete a range of adipokines that exhibit distinct biological functions. Here, we sought to understand the secretory pathways utilised by ACRP30 to the surface of adipocytes. We find that ACRP30 overlaps with adipsin in intracellular compartments distinct from Glut4, but nonetheless exhibits insulin-stimulated secretion from cells. Both adipsin and ACRP30 overlap with transferrin receptor-positive membranes, implying that the pathway of secretion involves the transferrin receptor-positive endosomal system. Consistent with this, we show that ablation of endosomes significantly inhibited the secretion of ACRP30, as did treatment of cells with Brefeldin A. In order to further probe the role of recycling endosomes on the secretion of ACRP30, we over-expressed a mutant form of Rab11, Rab11-S25N, in 3T3-L1 adipocytes and found that expression of this mutant significantly reduced basal and insulin-stimulated secretion. We also demonstrate that Arf6 also plays a role in the secretion of ACRP30. Collectively, these data implicate both Arf6 and Rab11 as crucial mediators of constitutive and insulin-stimulated secretion of ACRP30 and further suggest that recycling endosomes may play a central role in this process.

Evidence for a role of the exocyst in insulin-stimulated Glut4 trafficking in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in adipocytes and muscle by inducing the redistribution of Glut4 from intracellular locations to the plasma membrane. The fusion of Glut4-containing vesicles at the plasma membrane is known to involve the target SNAREs syntaxin 4 and SNAP-23 and the vesicle SNARE VAMP2. Little is known about the initial docking of Glut4 vesicles with the plasma membrane. A recent report has implicated Exo70, a component of the mammalian exocyst complex, in the initial interaction of Glut4 vesicles with the adipocyte plasma membrane. Here, we have examined the role of two other exocyst components, rsec6 and rsec8. We show that insulin promotes a redistribution of rsec6 and rsec8 to the plasma membrane and to cytoskeletal fractions within 3T3-L1 adipocytes but does not modulate levels of these proteins co-localized with Glut4. We further show that adenoviral-mediated overexpression of either rsec6 or rsec8 increases the magnitude of insulin-stimulated glucose transport in 3T3-L1 adipocytes. By contrast, overexpression of rsec6 or rsec8 did not increase the extent of the secretion of adipsin or ACRP30 from adipocytes and had no discernible effect on transferrin receptor traffic. Collectively, our data support a role for the exocyst in insulin-stimulated glucose transport and suggest a model by which insulin-dependent relocation of the exocyst to the plasma membrane may contribute to the specificity of Glut4 vesicle docking and fusion with the adipocyte plasma membrane.

Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes.

Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.