Summary Cytosolic DNA arising from intracellular bacteria or viral infections is a powerful pathogen-associated molecular pattern (PAMP) that leads to innate immune host defense by the production of type I interferon and inflammatory cytokines. Recognition of cytosolic DNA by the recently discovered cyclic-GMP-AMP (cGA) synthase (cGAS) induces the production of cGA to activate the stimulator of interferon genes (STING). Here we report the crystal structure of cGAS alone and in complex with DNA, ATP and GTP along with functional studies. Our results explain cGAS’ broad specificity DNA sensing, show how cGAS catalyzes di-nucleotide formation and indicate activation by a DNA-induced structural switch. cGAS possesses a remarkable structural similarity to the antiviral cytosolic dsRNA sensor 2’-5’oligoadenylate synthase (OAS1), but contains a unique zinc-thumb that recognizes B-form dsDNA. Our results mechanistically unify dsRNA and dsDNA innate immune sensing by OAS1 and cGAS nucleotidyl transferases.

The spindle checkpoint generates a ''wait anaphase'' signal at unattached kinetochores to prevent premature anaphase onset. Kinetochore-localized dynein is thought to silence the checkpoint by transporting checkpoint proteins from microtubule-attached kinetochores to spindle poles. Throughout metazoans, dynein recruitment to kinetochores requires the protein Spindly. Here, we identify a conserved motif in Spindly that is essential for kinetochore targeting of dynein. Spindly motif mutants, expressed following depletion of endogenous Spindly, target normally to kinetochores but prevent dynein recruitment. Spindly depletion and Spindly motif mutants, despite their similar effects on kinetochore dynein, have opposite consequences on chromosome alignment and checkpoint silencing. Spindly depletion delays chromosome alignment, but Spindly motif mutants ameliorate this defect, indicating that Spindly has a dynein recruitment-independent role in alignment. In Spindly depletions, the checkpoint is silenced following delayed alignment by a kinetochore dynein-independent mechanism. In contrast, Spindly motif mutants are retained on microtubule-attached kinetochores along with checkpoint proteins, resulting in persistent checkpoint signaling. Thus, dynein-mediated removal of Spindly from microtubuleattached kinetochores, rather than poleward transport per se, is the critical reaction in checkpoint silencing. In the absence of Spindly, a second mechanism silences the checkpoint; this mechanism is likely evolutionarily ancient, as fungi and higher plants lack kinetochore dynein. Microtubule attachments of the correct geometry are stabilized by tension experienced at sister kinetochores that have made bioriented connections to opposite poles (Nicklas 1997). Once all kinetochores are attached in a bioriented fashion to microtubule bundles, termed kinetochore fibers, the checkpoint signal is silenced and the cell proceeds to anaphase.The spindle checkpoint regulates the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which targets cyclin B and securin for destruction by the 26S proteasome. Specifically, the checkpoint components Mad2, BubR1, and Bub3 interact with and inhibit the essential APC/C cofactor Cdc20 by forming diffusible mitotic checkpoint complexes (Hwang et al. 1998;Sudakin et al. 2001;Nilsson et al. 2008). Additional components of the checkpoint pathway, including Mad1 and the kinases Bub1 and Mps1, are involved in the generation and amplification of the checkpoint signal (Hoyt et al. 1991;Li and Murray 1991;Abrieu et al. 2001).The conserved KNL-1/Mis12 complex/Ndc80 complex (KMN) network constitutes the core attachment site for microtubules at the kinetochore and also recruits components that generate the checkpoint signal (Burke and Stukenberg 2008). Additional contacts to microtubules are made by the kinesin CENP-E (Weaver et al. 2003) and by the minus end-directed motor dynein and its Cold Spring Harbor Laboratory Press on May 11, 2018 -Published by genesdev.cshlp.org Downloaded from

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill‐defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG‐I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG‐I‐dependent IL‐1β‐production and inflammasome activation. The signalling molecule CARD9 contributed to IL‐1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG‐I provides a mechanistic explanation for efficient induction of immunity by live bacteria.

The RZZ complex recruits dynein to kinetochores. We investigated structure, topology, and interactions of the RZZ subunits (ROD, ZWILCH, and ZW10) in vitro, in vivo, and in silico. We identify neuroblastoma-amplified gene (NAG), a ZW10 binder, as a ROD homolog. ROD and NAG contain an N-terminal beta propeller followed by an alpha solenoid, which is the architecture of certain nucleoporins and vesicle coat subunits, suggesting a distant evolutionary relationship. ZW10 binding to ROD and NAG is mutually exclusive. The resulting ZW10 complexes (RZZ and NRZ) respectively contain ZWILCH and RINT1 as additional subunits. The X-ray structure of ZWILCH, the first for an RZZ subunit, reveals a novel fold distinct from RINT1's. The evolutionarily conserved NRZ likely acts as a tethering complex for retrograde trafficking of COPI vesicles from the Golgi to the endoplasmic reticulum. The RZZ, limited to metazoans, probably evolved from the NRZ, exploiting the dynein-binding capacity of ZW10 to direct dynein to kinetochores.

RIG-I detects cytosolic viral dsRNA with 50 triphosphates (5 0 -pppdsRNA), thereby initiating an antiviral innate immune response. Here we report the crystal structure of superfamily 2 (SF2) ATPase domain of RIG-I in complex with a nucleotide analogue. RIG-I SF2 comprises two RecA-like domains 1A and 2A and a helical insertion domain 2B, which together form a 'C'-shaped structure. Domains 1A and 2A are maintained in a 'signal-off' state with an inactive ATP hydrolysis site by an intriguing helical arm. By mutational analysis, we show surface motifs that are critical for dsRNA-stimulated ATPase activity, indicating that dsRNA induces a structural movement that brings domains 1A and 2A/B together to form an active ATPase site. The structure also indicates that the regulatory domain is close to the end of the helical arm, where it is well positioned to recruit 5 0 -ppp-dsRNA to the SF2 domain. Overall, our results indicate that the activation of RIG-I occurs through an RNA-and ATP-driven structural switch in the SF2 domain.

The attachment of chromosomes to spindle microtubules during mitosis is a delicate and intricate process on which eukaryotic cells critically depend to maintain their ploidy. In this issue of Genes & Development, Gassmann and colleagues (pp. 2385-2399) present an analysis of the recently discovered Spindly/SPDL-1 protein that casts new lights onto the attachment process and the way it relates to the control of cell cycle progression. The basicsMitotic prometaphase is quite an eventful phase of the eukaryotic cell cycle. Its most characteristic trait is the mitotic spindle's frantic engagement in the capture of replicated chromosomes (the sister chromatids) that have been scattered throughout the cell. Microtubules, the main ingredient of the mitotic spindle, form tight attachments with specialized structures on mitotic chromosomes known as kinetochores. The correct configuration of kinetochore-microtubule attachment is named biorientation (Fig. 1A). When bioriented, the chromatids in a sister chromatid pair in the mother cell are connected to opposite spindle poles (Cheeseman and Desai 2008;Tanaka and Desai 2008). Biorientation contributes to chromosome congression to the metaphase plate. It also ensures that when the cohesion linking the sisters is removed at the metaphase-to-anaphase transition, the sisters are separated toward opposite spindle poles to give rise to two daughter cells with identical genetic material (Fig. 1B). The kinetochoreThe 60-80 conserved proteins that populate mitotic kinetochores from yeast to humans can be schematically subdivided into distinct functional modules (for review, see Cheeseman and Desai 2008). The first module is implicated in the interaction of kinetochores with centromeric chromatin, and is built around a specialized nucleosome containing the histone H3 variant CENP-A and associated binding partners in the so-called constitutive centromere-associated network (CCAN; also known as NAC/CAD) (Cheeseman and Desai 2008). The second module provides the core of the microtubulebinding interface. Its most prominent component is the KNL1-Mis12-Ndc80 complex (KMN) network, an array of 10 proteins (Cheeseman and Desai 2008). Besides creating a receptor for the microtubule, the KMN network also serves as a recruitment pad for additional proteins, including molecular motors like dynein, which have been implicated in the early stages of attachment (see below). Although the points of contact between the first and second modules have not been elucidated, the two modules contribute a structural core of the kinetochore that physically links chromosomes to spindle microtubules (Fig. 1A).The additional modules are implicated in the control of the state of kinetochore-microtubule attachment. One module includes the components of the spindle assembly checkpoint (SAC), which are all recruited to kinetochores in mitosis (Musacchio and Salmon 2007). The SAC alleviates the potentially hazardous consequences of attachment being largely (although not solely) based on the random encounter of a kineto...

RIG-I is a key pattern recognition receptor that recognizes cytoplasmic viral RNA. Upon ligand binding, it undergoes a conformational change that induces an active signaling conformation. However, the details of this conformational change remain elusive until high-resolution crystal structures of different functional conformations are available. X-ray crystallography is a powerful tool to study structure-function relationships, but crystallization is often the limiting step of the method. Here, we describe the in situ in-drop proteolysis of RIG-I that yielded crystals of the ATPase domain of mouse RIG-I suitable for structure determination.

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