The mtDNA mutation spectrum of the progeroid Polg mutator mouse includes abundant control region multimers

Polg mtDNA mutator mice are important models for investigating the role of acquired mtDNA mutations in aging. Despite extensive study, there remains little consensus on either the etiology of the progeroid phenotype or the mtDNA mutation spectrum induced by disrupted polymerase-γ function. To investigate the latter, we have developed a novel, pragmatic approach we term "Mito-seq," applying next-generation sequencing to enriched, native mtDNA. Regardless of detection parameters we observed an increase of at least two orders of magnitude in the number of mtDNA single nucleotide variants in Polg mutator mice compared to controls. We found no evidence for the accumulation of canonical mtDNA deletions but multimers of the mtDNA control region were identified in brain and heart. These control region multimers (CRMs) contained heterogeneous breakpoints and formed species that excluded the majority of mtDNA genes. CRMs demonstrate that polymerase-γ 3'-5' exonuclease activity is required for preserving mtDNA integrity.     

The length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis

Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV), an enveloped virus with bullet-shaped virions that measure 70 x 200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long) defective interfering particle, DI-T. By imaging the uptake of individual particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognition properties of a ligand are maintained.     

Comparison of micrometer‐ and scanning electron microscope‐based measurements of avian eggshell thickness

ABSTRACT The study of avian eggshell structure, including composition, pigmentation, thickness, and strength, has important ecological and economic implications. Previous investigators have used a variety of techniques to derive either direct measures or indirect estimates of eggshell thickness. Assessing the repeatability and method agreement of different techniques is necessary to permit comparison of eggshell thickness values from different studies on various genetic stocks, populations, and species. We recorded and analyzed measurements of eggshell thickness using two methods, micrometers and scanning electron microscopy (SEM), for several Palaeognathae and Neognathae taxa, including nonpasserines and passerines. Applying a tolerance‐interval approach, we found that repeatability of measurements for eggs with thinner shells (<300 μm, all Neognathae taxa) was worse than for eggs with thicker shells (Palaeognathae taxa), but was still statistically and biologically reasonable given that the relative magnitude of intramethod agreements was <11%. Our results support previous predictions that measurements made using a micrometer are comparable to those made using SEM. This finding is particularly important given the relative ease and cost efficiency of the micrometer method. Importantly, these new analyses can be used to validate the use of published data from previous studies of micrometer‐based eggshell thickness for both intra‐ and interspecific comparisons.     

Trex1 exonuclease degrades ssDNA to prevent chronic checkpoint activation and autoimmune disease

Trex1 is the major 3' DNA exonuclease in mammalian cells, and mutations in the human TREX1 gene can cause Aicardi-Goutières syndrome, characterized by perturbed immunity. Similarly, Trex1(-/-) mice have an autoinflammatory phenotype; however, the mechanism of Trex1-deficient disease is unknown. We report that Trex1, ordinarily associated with the endoplasmic reticulum (ER), relocalizes to the S phase nucleus after gamma irradiation or hydroxyurea treatment. Notably, Trex1-deficient cells show defective G1/S transition and chronic ATM-dependent checkpoint activation, even in the absence of exogenous stress, correlating with persistent single-stranded DNA molecules produced in S phase, which accumulate in the ER. Our data indicate that Trex1 acts on a single-stranded DNA polynucleotide species generated from processing of aberrant replication intermediates to attenuate DNA damage checkpoint signaling and prevent pathological immune activation.     

Structural basis for the inhibition of tyrosine kinase activity of ZAP-70

ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.     

The likely impact of elevated [CO2], nitrogen deposition, increased temperature and management on carbon sequestration in temperate and boreal forest ecosystems: a literature review

Temperate and boreal forest ecosystems contain a large part of the carbon stored on land, in the form of both biomass and soil organic matter. Increasing atmospheric [CO2], increasing temperature, elevated nitrogen deposition and intensified management will change this C store. Well documented single-factor responses of net primary production are: higher photosynthetic rate (the main [CO2] response); increasing length of growing season (the main temperature response); and higher leaf-area index (the main N deposition and partly [CO2] response). Soil organic matter will increase with increasing litter input, although priming may decrease the soil C stock initially, but litter quality effects should be minimal (response to [CO2], N deposition, and temperature); will decrease because of increasing temperature; and will increase because of retardation of decomposition with N deposition, although the rate of decomposition of high-quality litter can be increased and that of low-quality litter decreased. Single-factor responses can be misleading because of interactions between factors, in particular those between N and other factors, and indirect effects such as increased N availability from temperature-induced decomposition. In the long term the strength of feedbacks, for example the increasing demand for N from increased growth, will dominate over short-term responses to single factors. However, management has considerable potential for controlling the C store.     

Up or down in space? Uniting the bottom‐up versus top‐down paradigm and spatial ecology

Why is the World green – what keeps herbivores, and herbivorous insects in particular, from consuming all of their food? When this question was first posed, the relative importance of top‐down and bottom‐up effects was hotly disputed. While modern ecologists may agree that impacts from several different directions will affect local insect densities, the bottom‐up vs top‐down jargon seems to be stuck in a unidimensional world. Here, we argue that the strength of almost every bottom‐up and top‐down force is likely to vary in space, and that in itself, spatial structure invokes new processes which defy classification in the traditional bottom‐up top‐down scheme. To understand the relative importance of different forces keeping herbivore numbers in check, we feel that we need a fresh synthesis between the novel paradigm of spatial ecology and the classical paradigms of top‐down and bottom‐up studies. This synthesis requires a consideration of forces beyond the standard framework of top‐down vs bottom‐up effects, and should be based on comparing the relative strength of such forces at several sites in a spatially explicit framework. Overall, we should switch our focus from whether the relative strength of top‐down and bottom‐up factors vary in space to why there is variation, how much variation there is, and at what spatial scale(s) it occurs.     

Food web structure and interaction strength pave the way for vulnerability to extinction

This paper focuses on how food web structure and interactions among species affects the vulnerability, due to environmental variability, to extinction of species at different positions in model food webs. Vulnerability is here not measured by a traditional extinction threshold but is instead inspired by the IUCN criteria for endangered species: an observed rapid decline in population abundance. Using model webs influenced by stochasticity with zero autocorrelation, we investigate the ecological determinants of species vulnerability, i.e. the trophic interactions between species and food web structure and how these interact with the risk of sudden drops in abundance of species. We find that (i) producers fulfil the criterion of vulnerable species more frequently than other species, (ii) food web structure is related to vulnerability, and (iii) the vulnerability of species is greater when involved in a strong trophic interaction than when not. We note that our result on the relationship between extinction risk and trophic position of species contradict previous suggestions and argue that the main reason for the discrepancy probably is due to the fact that we study the vulnerability to environmental stochasticity and not extinction risk due to overexploitation, habitat destruction or interactions with introduced species. Thus, we suggest that the vulnerability of species to environmental stochasticity may be differently related to trophic position than the vulnerability of species to other factors. Earlier research on species extinctions has looked for intrinsic traits of species that correlate with increased vulnerability to extinction. However, to fully understand the extinction process we must also consider that species interactions may affect vulnerability and that not all extinctions are the result of long, gradual reductions in species abundances. Under environmental stochasticity (which importance frequently is assumed to increase as a result of climate change) and direct and indirect interactions with other species some extinctions may occur rapidly and apparently unexpectedly. To identify the first declines of population abundances that may escalate and lead to extinctions as early as possible, we need to recognize which species are at greatest risk of entering such dangerous routes and under what circumstances. This new perspective may contribute to our understanding of the processes leading to extinction of populations and eventually species. This is especially urgent in the light of the current biodiversity crisis where a large fraction of the world's biodiversity is threatened.