SUMMARY RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

SUMMARY Mouse p202 containing two HIN domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found only HIN1 of p202 binds dsDNA, while HIN2 forms a homo-tetramer. Crystal structures of HIN1 revealed that dsDNA is bound on the opposite face to the site used in AIM2 and IFI16. The structure of HIN2 revealed a dimer of dimers, with the face analogous to the HIN1 dsDNA binding site being a dimerization interface. Electron microscopy imaging showed that HIN1 is flexibly linked to HIN2 in p202, and tetramerization provided enhanced avidity for dsDNA. Surprisingly, HIN2 of p202 interacts with AIM HIN domain. We propose this results in spatial separation of AIM2 pyrin domains, and indeed p202 prevented dsDNA-dependent clustering of ASC and AIM2 inflammasome activation. We hypothesize that while p202 was evolutionarily selected to limit AIM2-mediated inflammation in some mouse strains, the same mechanism contributes to increased interferon production and lupus susceptibility.

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3eγ, CD3eδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3eδ complex reveals the CD3eδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3eδ and CD3eγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.T-cell receptor | electron microscopy | small-angle X-ray scattering T cells are key mediators of the adaptive immune response.Each αβ T cell contains a unique αβ T-cell receptor (TCR), which binds antigens (Ags) displayed by major histocompatibility complexes (MHCs) and MHC-like molecules (1). The TCR serves as a remarkably sensitive driver of cellular function: although TCR ligands typically bind quite weakly (1-200 μM), even a handful of TCR ligands are sufficient to fully activate a T cell (2, 3). The TCR does not possess intracellular signaling domains, uncoupling Ag recognition from T-cell signaling. The TCR is instead noncovalently associated with a multisubunit signaling apparatus, consisting of the CD3eγ and CD3eδ heterodimers and the CD3ζζ homodimer, which collectively form the TCR-CD3 complex (4, 5). The CD3γ/δ/e subunits each consist of a single extracellular Ig domain and a single immunoreceptor tyrosine-based activation motif (ITAM), whereas CD3ζ has a short extracellular domain (ECD) and three ITAMs (6-11). The TCR-CD3 complex exists in 1:1:1:1 stoichiometry for the αβTCR: CD3eγ:CD3eδ:CD3ζζ dimers (12). Phosphorylation of the intracellular CD3 ITAMs and recruitment of the adaptor Nck lead to T-cell activation, proliferation, and survival (13,14). Understanding the underlying principles of TCR-CD3 architecture and T-cell signaling is of therapeutic interest. For example, TCR-CD3 is the target of therapeutic antibodies such as the immunosuppressant OKT3 (15), and there is increasing interest in manipulating T cells in an Ag-dependent manner by using naturally occurring and engineered TCRs (16).Assembly of the TCR-CD3 complex is p...

Circulating fetal nucleated cells (CFNCs) in maternal blood offer an ideal source of fetal genomic DNA for noninvasive prenatal diagnostics (NIPD). We developed a class of nanoVelcro microchips to effectively enrich a subcategory of CFNCs, i.e., circulating trophoblasts (cTBs) from maternal blood, which can then be isolated with single-cell resolution by a laser capture microdissection (LCM) technique for downstream genetic testing. We first established a nanoimprinting fabrication process to prepare the LCM-compatible nanoVelcro substrates. Using an optimized cTB-capture condition and an immunocytochemistry protocol, we were able to identify and isolate single cTBs (Hoechst+/CK7+/HLA-G+/CD45−, 20 μm > sizes > 12 μm) on the imprinted nanoVelcro microchips. Three cTBs were polled to ensure reproducible whole genome amplification on the cTB-analysis. Using maternal blood samples collected from expectant mothers carrying a single fetus, the cTB-derived aCGH data were able to detect fetal genders and chromosomal aberrations, which had been confirmed by standard clinical practice. Our results support the use of nanoVelcro microchips for cTB-based noninvasive prenatal genetic testing, which holds potential for further development toward future NIPD solution.

In this study, we devised a simple method to enhance the conductivity of poly(3,4ethylenedioxythiophene)-poly(styrene-sulfonate) (PEDOT:PSS) films through spin-coating with various surface-modified compounds, and then applied this technique to the preparation of ITO-free polymer solar cells (PSCs). The electrical conductivity of PEDOT:PSS films can be increased by more than two order of magnitudes merely by spin-coating a compound containing one or more polar groups-such as ethanol, methoxyethanol, 1,2-dimethoxyethane, and ethylene glycol-onto the films. In this paper, we discuss the phenomena occurring through conductivities, morphologies, and chemical properties of the modified PEDOT-PSS films as determined using Raman spectroscopy, a four-point probe, scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). The schematic 3D morphological model of directly solvent-modified PEDOT:PSS films is presumed for ITO-free devices. The desirable conductivity enhancements of these materials make them attractive candidates for use as anode materials in ITO-free PSCs.