Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

The Spindle Assembly Checkpoint (SAC) is a unique signaling mechanism that responds to the state of attachment of the kinetochore to spindle microtubules. SAC signaling is activated by unattached kinetochores, and it is silenced after these kinetochores form end-on microtubule attachments. Although the biochemical cascade of SAC signaling is well-understood, how kinetochore-microtubule attachment disrupts it remained unknown. Here we show that, in budding yeast, end-on microtubule attachment to the kinetochore physically separates the Mps1 kinase, which likely binds to the Calponin homology domain of Ndc80, from the kinetochore substrate of Mps1, Spc105 (KNL1 orthologue). This attachment-mediated separation disrupts the phosphorylation of Spc105, and enables SAC silencing. Additionally, the Dam1 complex may act as a barrier that shields Spc105 from Mps1. Together these data suggest that the protein architecture of the kinetochore encodes a mechanical switch. End-on microtubule attachment to the kinetochore turns this switch off to silence the SAC.

The spliceosome is a complex small nuclear (sn)RNA-protein machine that removes introns from pre-mRNAs via two successive phosphoryl transfer reactions. The chemical steps are isoenergetic, yet splicing requires at least eight RNA-dependent ATPases responsible for substantial conformational rearrangements. To comprehensively monitor pre-mRNA conformational dynamics, we developed a strategy for single molecule FRET (smFRET) that utilizes a small, efficiently spliced yeast pre-mRNA, Ubc4, in which donor and acceptor fluorophores are placed in the exons adjacent to the 5′ and 3′ splice sites. During splicing in vitro we observe a multitude of generally reversible, time-and ATP-dependent conformational transitions of individual pre-mRNAs. The conformational dynamics of branchpoint and 3′ splice site mutants differ from one another and from wild-type. Because all transitions are reversible, spliceosome assembly appears to be occurring close to thermal equilibrium.

Background The kinetochore is a multiprotein machine that couples chromosome movement to microtubule (MT) polymerization and depolymerization. It uses numerous copies of at least three MT-binding proteins to generate bidirectional movement. The nanoscale organization of these proteins within the kinetochore plays an important role in shaping the mechanisms that drive persistent, bidirectional movement of the kinetochore. Results We used Förster Resonance Energy Transfer (FRET) between genetically encoded fluorescent proteins fused to kinetochore subunits to reconstruct the nanoscale organization of the budding yeast kinetochore. We performed > 60 FRET and high resolution colocalization measurements involving the essential MT-binding kinetochore components: Ndc80, Dam1, Spc105, and Stu2. These measurements reveal that neighboring Ndc80 complexes within the kinetochore are narrowly distributed along the length of the MT. Dam1 complex molecules are concentrated near the MT-binding domains of Ndc80. Stu2 localizes in high abundance within a narrowly defined territory within the kinetochore centered ~ 20 nm on the centromeric side of the Dam1 complex. Conclusions Our data show that the microtubule attachment site of the budding yeast kinetochore is well-organized. Ndc80, Dam1 and Stu2 are all narrowly distributed about their average positions along the kinetochore-MT axis. The relative organization of these components, their narrow distributions, and their known MT-binding properties together elucidate how their combined actions generate persistent, bidirectional kinetochore movement coupled to MT polymerization and depolymerization.