Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions

The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.     

Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance

Recognition of self-antigen-derived epitopes presented by major histocompatibility complex class II (MHC II) molecules on thymic epithelial cells (TECs) is critical for the generation of a functional and self-tolerant CD4 T-cell repertoire. Whereas haematopoietic antigen-presenting cells generate MHC-II-peptide complexes predominantly through the processing of endocytosed polypeptides, it remains unknown if and how TECs use unconventional pathways of antigen presentation. Here we address the role of macroautophagy, a process that has recently been shown to allow for endogenous MHC II loading, in T-cell repertoire selection in the mouse thymus. In contrast to most other tissues, TECs had a high constitutive level of autophagy. Genetic interference with autophagy specifically in TECs led to altered selection of certain MHC-II-restricted T-cell specificities and resulted in severe colitis and multi-organ inflammation. Our findings indicate that autophagy focuses the MHC-II-peptide repertoire of TECs on their intracellular milieu, which notably comprises a wide array of otherwise strictly 'tissue-specific' self antigens. In doing so, it contributes to T-cell selection and is essential for the generation of a self-tolerant T-cell repertoire.     

Endocytosis and inositol hexakisphosphate levels in ras transformants of Dictyostelium discoideum amoebae

Fluid-phase pinocytosis kinetics and lysosomal enzyme secretion parameters were measured in Dictyostelium discoideum amoebae constructed from strain AX3 by transformation with a multicopy plasmid carrying either a normal ras gene (ras-Gly12), a mutated ras gene (ras-Thr12) or by the vector carrying the geneticin resistance gene only (pDNEO2). It was found that the pinocytosis rate and extent as well as the lysosomal enzyme secretion were slightly different in the three strains. These changes, however, were related to minor modifications of the cellular volumes. The overall concentration of inositol hexakisphosphate was similar in the three strains.     

Proteome survey reveals modularity of the yeast cell machinery

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.     

Fluidity of water confined to subnanometre films.

The fluidity of water in confined geometries is relevant to processes ranging from tribology to protein folding, and its molecular mobility in pores and slits has been extensively studied using a variety of approaches. Studies in which liquid flow is measured directly suggest that the viscosity of aqueous electrolytes confined to films of thickness greater than about 2-3 nm remains close to that in the bulk; this behaviour is similar to that of non-associative organic liquids confined to films thicker than about 7-8 molecular layers. Here we observe that the effective viscosity of water remains within a factor of three of its bulk value, even when it is confined to films in the thickness range 3.5 +/- 1 to 0.0 +/- 0.4 nm. This contrasts markedly with the behaviour of organic solvents, whose viscosity diverges when confined to films thinner than about 5-8 molecular layers. We attribute this to the fundamentally different mechanisms of solidification in the two cases. For non-associative liquids, confinement promotes solidification by suppressing translational freedom of the molecules; however, in the case of water, confinement seems primarily to suppress the formation of the highly directional hydrogen-bonded networks associated with freezing.     

Germline KRAS mutations cause Noonan syndrome

Noonan syndrome (MIM 163950) is characterized by short stature, facial dysmorphism and cardiac defects. Heterozygous mutations in PTPN11, which encodes SHP-2, cause approximately 50% of cases of Noonan syndrome. The SHP-2 phosphatase relays signals from activated receptor complexes to downstream effectors, including Ras. We discovered de novo germline KRAS mutations that introduce V14I, T58I or D153V amino acid substitutions in five individuals with Noonan syndrome and a P34R alteration in a individual with cardio-facio-cutaneous syndrome (MIM 115150), which has overlapping features with Noonan syndrome. Recombinant V14I and T58I K-Ras proteins show defective intrinsic GTP hydrolysis and impaired responsiveness to GTPase activating proteins, render primary hematopoietic progenitors hypersensitive to growth factors and deregulate signal transduction in a cell lineage-specific manner. These studies establish germline KRAS mutations as a cause of human disease and infer that the constellation of developmental abnormalities seen in Noonan syndrome spectrum is, in large part, due to hyperactive Ras.