Invariant NKT cells require autophagy to coordinate proliferation and survival signals during differentiation.

Autophagy regulates cell differentiation, proliferation, and survival in multiple cell types, including cells of the immune system. In this study, we examined the effects of a disruption of autophagy on the differentiation of invariant NKT (iNKT) cells. Using mice with a T lymphocyte-specific deletion of Atg5 or Atg7, two members of the macroautophagic pathway, we observed a profound decrease in the iNKT cell population. The deficit is cell-autonomous, and it acts predominantly to reduce the number of mature cells, as well as the function of peripheral iNKT cells. In the absence of autophagy, there is reduced progression of iNKT cells in the thymus through the cell cycle, as well as increased apoptosis of these cells. Importantly, the reduction in Th1-biased iNKT cells is most pronounced, leading to a selective reduction in iNKT cell-derived IFN-γ. Our findings highlight the unique metabolic and genetic requirements for the differentiation of iNKT cells.

Autophagy is essential for effector CD8(+) T cell survival and memory formation.

The importance of autophagy in the generation of memory CD8(+) T cells in vivo is not well defined. We report here that autophagy was dynamically regulated in virus-specific CD8(+) T cells during acute infection of mice with lymphocytic choriomeningitis virus. In contrast to the current paradigm, autophagy decreased in activated proliferating effector CD8(+) T cells and was then upregulated when the cells stopped dividing just before the contraction phase. Consistent with those findings, deletion of the gene encoding either of the autophagy-related molecules Atg5 or Atg7 had little to no effect on the proliferation and function of effector cells, but these autophagy-deficient effector cells had survival defects that resulted in compromised formation of memory T cells. Our studies define when autophagy is needed during effector and memory differentiation and warrant reexamination of the relationship between T cell activation and autophagy.

Nondegradative role of Atg5-Atg12/ Atg16L1 autophagy protein complex in antiviral activity of interferon gamma.

Host resistance to viral infection requires type I (α/ß) and II (γ) interferon (IFN) production. Another important defense mechanism is the degradative activity of macroautophagy (herein autophagy), mediated by the coordinated action of evolutionarily conserved autophagy proteins (Atg). We show that the Atg5-Atg12/Atg16L1 protein complex, whose prior known function is in autophagosome formation, is required for IFNγ-mediated host defense against murine norovirus (MNV) infection. Importantly, the direct antiviral activity of IFNγ against MNV in macrophages required Atg5-Atg12, Atg7, and Atg16L1, but not induction of autophagy, the degradative activity of lysosomal proteases, fusion of autophagosomes and lysosomes, or the Atg8-processing protein Atg4B. IFNγ, via Atg5-Atg12/Atg16L1, inhibited formation of the membranous cytoplasmic MNV replication complex, where Atg16L1 localized. Thus, the Atg5-Atg12/Atg16L1 complex performs a pivotal, nondegradative role in IFNγ-mediated antiviral defense, establishing that multicellular organisms have evolved to use portions of the autophagy pathway machinery in a cassette-like fashion for host defense.

Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants.

We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNA(Ser)UCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNA(Ser)UCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNA(Ser)UCA-C47:6U levels in sla1-rrm but not sla1-null cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a 'reference' mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.

Bone morphogenetic proteins signal through the transforming growth factor-beta type III receptor.

The bone morphogenetic protein (BMP) family, the largest subfamily of the structurally conserved transforming growth factor-beta (TGF-beta) superfamily of growth factors, are multifunctional regulators of development, proliferation, and differentiation. The TGF-beta type III receptor (TbetaRIII or betaglycan) is an abundant cell surface proteoglycan that has been well characterized as a TGF-beta and inhibin receptor. Here we demonstrate that TbetaRIII functions as a BMP cell surface receptor. TbetaRIII directly and specifically binds to multiple members of the BMP subfamily, including BMP-2, BMP-4, BMP-7, and GDF-5, with similar kinetics and ligand binding domains as previously identified for TGF-beta. TbetaRIII also enhances ligand binding to the BMP type I receptors, whereas short hairpin RNA-mediated silencing of endogenous TbetaRIII attenuates BMP-mediated Smad1 phosphorylation. Using a biologically relevant model for TbetaRIII function, we demonstrate that BMP-2 specifically stimulates TbetaRIII-mediated epithelial to mesenchymal cell transformation. The ability of TbetaRIII to serve as a cell surface receptor and mediate BMP, inhibin, and TGF-beta signaling suggests a broader role for TbetaRIII in orchestrating TGF-beta superfamily signaling.

The type III TGF-beta receptor signals through both Smad3 and the p38 MAP kinase pathways to contribute to inhibition of cell proliferation.

Transforming growth factor beta (TGFbeta) has an important role as a negative regulator of cellular proliferation. The type III transforming growth factor beta receptor (TbetaRIII) has an emerging role as both a TGFbeta superfamily co-receptor and in mediating signaling through its cytoplasmic domain. In L6 myoblasts, TbetaRIII expression enhanced TGFbeta1-mediated growth inhibition, with this effect mediated, in part, by the TbetaRIII cytoplasmic domain. The effects of TbetaRIII were not due to altered ligand presentation or to differences in Smad2 phosphorylation. Instead, TbetaRIII specifically increased Smad3 phosphorylation, both basal and TGFbeta-stimulated Smad3 nuclear localization and Smad3-dependent activation of reporter genes independent of its cytoplasmic domain. Conversely, SB431542, a type I transforming growth factor beta receptor (TbetaRI) inhibitor, as well as dominant-negative Smad3 specifically and significantly abrogated the effects of TbetaRIII on TGFbeta1-mediated inhibition of proliferation. TbetaRIII also specifically increased p38 phosphorylation, and SB203580, a p38 kinase inhibitor, specifically and significantly abrogated the effects of TbetaRIII/TGFbeta1-mediated inhibition of proliferation in L6 myoblasts and in primary human epithelial cells. Importantly, treatment with the TbetaRI and p38 inhibitors together had additive effects on abrogating TbetaRIII/TGFbeta1-mediated inhibition of proliferation. In a reciprocal manner, short hairpin RNA-mediated knockdown of endogenous TbetaRIII in various human epithelial cells attenuated TGFbeta1-mediated inhibition of proliferation. Taken together, these data demonstrate that TbetaRIII contributes to and enhances TGFbeta-mediated growth inhibition through both TbetaRI/Smad3-dependent and p38 mitogen-activated protein kinase pathways.

The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells.

The La protein is a target of autoantibodies in patients suffering from Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La+/-intercrosses were analyzed. La-/- offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La+/- intercrosses yielded 38 La+/+ and La+/- embryonic stem (ES) cell lines but no La-/- ES cell lines, suggesting that La contributes a critical function toward the establishment or survival of ES cells. Consistent with this, La-/- blastocyst outgrowths revealed loss of the inner cell mass (ICM). The results indicate that in contrast to the situation in yeasts, La is essential in mammals and is one of a limited number of genes required as early as the development of the ICM.