Cellular senescence correlates with changes in the transcriptome. To obtain a complete view on senescence-associated transcription networks and pathways, we assessed by deep RNA sequencing the ...transcriptomes of five of the most commonly used laboratory strains of human fibroblasts during their transition into senescence. In a number of cases, we verified the RNA-seq data by real-time PCR. By determining cellular protein levels we observed that the age-related expression of most but not all genes is regulated at the transcriptional level. We found that 78% of the age-affected differentially expressed genes were commonly regulated in the same direction (either up- or down-regulated) in all five fibroblast strains, indicating a strong conservation of age-associated changes in the transcriptome. KEGG pathway analyses confirmed up-regulation of the senescence-associated secretory phenotype and down-regulation of DNA synthesis/repair and most cell cycle pathways common in all five cell strains. Newly identified senescence-induced pathways include up-regulation of endocytotic/phagocytic pathways and down-regulation of the mRNA metabolism and the mRNA splicing pathways. Our results provide an unprecedented comprehensive and deep view into the individual and common transcriptome and pathway changes during the transition into of senescence of five human fibroblast cell strains.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The biophysical mechanism of liquid-liquid phase separation (LLPS) has emerged as an attractive idea to explain the formation and function of membrane-less organelles, such as nuclear bodies. Our aim ...is to simulate the genesis of promyelocytic leukemia nuclear bodies (PML-NBs) at the molecular level using computer models in order to gain insight on LLPS within PML-NBs. To this end, we have generated a computer model of PML-NB assembly which uncovers molecular details of their genesis and a spatial map of its LLPS-driving elements. Model predictions can be exploited to test new hypotheses of PML-NB structure in the wet lab in an iterative process. The established computer model may be expanded to other membrane-less organelles to reveal their structural details.
The paper describes the Rosetta Lander named Philae and introduces its complement of scientific instruments. Philae was launched aboard the European Space Agency Rosetta spacecraft on 02 March 2004 ...and is expected to land and operate on the nucleus of 67P/Churyumov-Gerasimenko at a distance of about 3 AU from the Sun. Its overall mass is ~98 kg (plus the support systems remaining on the Orbiter), including its scientific payload of ~27 kg. It will operate autonomously, using the Rosetta Orbiter as a communication relay to Earth. The scientific goals of its experiments focus on elemental, isotopic, molecular and mineralogical composition of the cometary material, the characterization of physical properties of the surface and subsurface material, the large-scale structure and the magnetic and plasma environment of the nucleus. In particular, surface and sub-surface samples will be acquired and sequentially analyzed by a suite of instruments. Measurements will be performed primarily during descent and along the first five days following touch-down. Philae is designed to also operate on a long time-scale, to monitor the evolution of the nucleus properties. Philae is a very integrated project at system, science and management levels, provided by an international consortium. The Philae experiments have the potential of providing unique scientific outcomes, complementing by in situ ground truth the Rosetta Orbiter investigations.
Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder selectively affecting axons of spinal cord motoneurons. Classical mutations in the most frequent HSP gene SPAST (spastin protein) ...act through haploinsufficiency by abolishing the activity of a C‐terminal ATPase domain or by interfering with expression from the affected allele. N‐terminal missense variants have been suggested to represent rare polymorphisms, to cause unusually mild phenotypes, and to aggravate the effect of a classical mutation. We confirm these associations for p.S44L but do not detect two other variants (p.E43Q; p.P45Q) in HSP patients and controls. We show that neither of several disease mechanisms associated with classical SPAST mutations applies to the N‐terminal variants. Instead, all three alterations enhance the stability of one of two alternative spastin isoforms. Their phenotypic effect may thus not be mediated by haploinsufficiency but by increasing isoform competition for interacting proteins, substrates or oligomerization partners.
The authors report a nucleotide substitution (c.1216A>G) in SPG4 (spastin) causing hereditary spastic paraplegia. This apparent missense mutation in the ATPase domain confers aberrant, in-frame ...splicing and results in destabilization of mutated transcript. Mutated protein is deficient in microtubule-severing activity but, unlike neighboring mutations, shows regular subcellular localization. The authors' data point to haploinsufficiency rather than a dominant negative effect as the disease-causing mechanism for this mutation.
Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein with functions in euchromatin and heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms in ...mammalian cells. Using fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) we found that in interphase cells most HP1 molecules (50-80%) are highly mobile (recovery halftime: t(1/2) approximately 0.9 s; diffusion coefficient: D approximately 0.6-0.7 microm(2) s(-1)). Twenty to 40% of HP1 molecules appear to be incorporated into stable, slow-moving oligomeric complexes (t(1/2) approximately 10 s), and constitutive heterochromatin of all mammalian cell types analyzed contain 5-7% of very slow HP1 molecules. The amount of very slow HP1 molecules correlated with the chromatin condensation state, mounting to more than 44% in condensed chromatin of transcriptionally silent cells. During mitosis 8-14% of GFP-HP1alpha, but not the other isoforms, are very slow within pericentromeric heterochromatin, indicating an isoform-specific function of HP1alpha in heterochromatin of mitotic chromosomes. These data suggest that mobile as well as very slow populations of HP1 may function in concert to maintain a stable conformation of constitutive heterochromatin throughout the cell cycle.
Formation of γ-H2AX foci is a P. O.cellular response to genotoxic stress, such as DNA double strand breaks or stalled replication forks. Here we show that γ-H2AX foci were also formed when cells were ...incubated with 0.5 μg/ml DNA intercalating agent actinomycin D. In untreated cells, γ-H2AX co-immunoprecipitated with Ku70, a subunit of DNA-dependent protein kinase, as well as with nuclear DNA helicase II (NDH II), a DEXH family helicase also known as RNA helicase A or DHX9. This association was increased manifold after actinomycin D treatment. DNA degradation diminished the amount of Ku70 associated with γ-H2AX but not that of NDH II. In vitro binding studies with recombinant NDH II and H2AX phosphorylated by DNA-dependent protein kinase confirmed a direct physical interaction between NDH II and γ-H2AX. Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to γ-H2AX. Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with γ-H2AX foci. Taken together, these results suggest that histone γ-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA.
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