Skeletal muscle weakness in osteogeneis imperfecta mice

Exercise intolerance, muscle fatigue and weakness are often-reported, little-investigated concerns of patients with osteogenesis imperfecta (OI). OI is a heritable connective tissue disorder hallmarked by bone fragility resulting primarily from dominant mutations in the proα1(I) or proα2(I) collagen genes and the recently discovered recessive mutations in post-translational modifying proteins of type I collagen. In this study we examined the soleus (S), plantaris (P), gastrocnemius (G), tibialis anterior (TA) and quadriceps (Q) muscles of mice expressing mild (+/oim) and moderately severe (oim/oim) OI for evidence of inherent muscle pathology. In particular, muscle weight, fiber cross-sectional area (CSA), fiber type, fiber histomorphology, fibrillar collagen content, absolute, relative and specific peak tetanic force (P_o, P_o/mg and P_o/CSA respectively) of individual muscles were evaluated. Oim/oim mouse muscles were generally smaller, contained less fibrillar collagen, had decreased P_o and an inability to sustain P_o for the 300-ms testing duration for specific muscles; +/oim mice had a similar but milder skeletal muscle phenotype. +/oim mice had mild weakness of specific muscles but were less affected than their oim/oim counterparts which demonstrated readily apparent skeletal muscle pathology. Therefore muscle weakness in oim mice reflects inherent skeletal muscle pathology.     

HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.     

Exome association analysis sheds light onto leaf rust (Puccinia triticina) resistance genes currently used in wheat breeding (Triticum aestivum L.)

Resistance breeding is crucial for a sustainable control of leaf rust (Puccinia triticina) in wheat (Triticum aestivum L.) while directly targeting functional variants is the Holy Grail for efficient marker‐assisted selection and map‐based cloning. We assessed the limits and prospects of exome association analysis for severity of leaf rust in a large hybrid wheat population of 1574 single‐crosses plus their 133 parents. After imputation and quality control, exome sequencing revealed 202 875 single‐nucleotide polymorphisms (SNPs) covering 19.7% of the high‐confidence annotated gene space. We performed intensive data mining and found significant associations for 2171 SNPs corresponding to 50 different loci. Some of these associations mapped in the proximity of the already known resistance genes Lr21, Lr34‐B, Lr1 and Lr10, while other associated genomic regions, such as those on chromosomes 1A and 3D, harboured several annotated genes putatively involved in resistance. Validation with an independent population helped to narrow down the list of putative resistance genes that should be targeted by fine‐mapping. We expect that the proposed strategy of intensive data mining coupled with validation will significantly influence research in plant genetics and breeding.     

Trophic niches of three sympatric invasive crayfish of EU concern

The spread of non-native species results in novel and often unexpected assemblages. Using stable isotopes, we disentangled the trophic relationships between three invasive crayfish species at two sites of a small thermal tributary of the Barát stream, Hungary. We studied Procambarus virginalis and Faxonius limosus living in sympatry in the upper section of this thermal tributary, and then an assemblage in a lower section also containing P. clarkii. The two species in the upper section largely shared trophic niches, although P. virginalis was more carnivorous than F. limosus, which fed more on detritus and aquatic plants. In the lower section, P. clarkii had a distinctive trophic niche, being more carnivorous than the other species and also preying on other crayfish and fish. The trophic niches of the other two species shifted slightly, being narrower and more overlapping in the presence of P. clarkii. It seems that the presence of P. clarkii affects the feeding habits and trophic niches of the other two crayfish. Our results also indicate that the species have somewhat distinctive feeding niches, which suggests that the ecosystem effects of these species are likely to be at least partially additive in the shared localities.

     

A Combined Approach of Quantitative Interaction Proteomics and Live-cell Imaging Reveals a Regulatory Role for Endoplasmic Reticulum (ER) Reticulon Homology Proteins in Peroxisome Biogenesis

Peroxisome biogenesis initiates at the endoplasmic reticulum (ER) and maturation allows for the formation of metabolically active organelles. Yet, peroxisomes can also multiply by growth and division. Several proteins, called peroxins, are known to participate in these processes but little is known about their organization to orchestrate peroxisome proliferation. Here, we demonstrate that regulation of peroxisome proliferation relies on the integrity of the tubular ER network. Using a dual track SILAC-based quantitative interaction proteomics approach, we established a comprehensive network of stable as well as transient interactions of the peroxin Pex30p, an integral membrane protein. Through association with merely ER resident proteins, in particular with proteins containing a reticulon homology domain, and with other peroxins, Pex30p designates peroxisome contact sites at ER subdomains. We show that Pex30p traffics through the ER and segregates in punctae to which peroxisomes specifically append, and we ascertain its transient interaction with all subunits of the COPI coatomer complex suggesting the involvement of a vesicle-mediated transport. We establish that the membrane protein Pex30p facilitates the connection of peroxisomes to the ER. Taken together, our data indicate that Pex30p-containing protein complexes act as focal points from which peroxisomes can form and that the tubular ER architecture organized by the reticulon homology proteins Rtn1p, Rtn2p and Yop1p controls this process.All nucleated cells contain essential round-shaped organelles called peroxisomes, whose function is mainly associated with lipid metabolism (1). Depending on the cellular requirements, the size, number, and protein content of these single membrane-bound organelles can vary widely. Although peroxisomes are dispensable for unicellular species such as yeasts, they are essential for the development of multicellular organisms (2, 3). In human, mutations in PEX genes lead to defects in peroxisome function or formation and are associated with the development of lethal pathologies (4). These PEX genes code for proteins, called peroxins, which are involved in peroxisome assembly and maintenance (5).Two major routes seem to lead to peroxisome formation, namely, de novo biogenesis and growth/division of pre-existing peroxisomes. The division pathway operates with proteins of the Pex11 family and requires fission factors shared with mitochondria (6). Studies in yeast and mammalian cells revealed that through the action of the protein Pex3p peroxisome precursors can also originate from the endoplasmic reticulum (ER)¹ and, via import of membrane and matrix proteins, mature into fully functional organelles (7, 8). Furthermore, several peroxisomal membrane proteins were shown to migrate to peroxisomes via the ER (7, 9, 10). The molecular mechanism underlying the biogenic pathway of peroxisome formation has not been clarified so far. Recent data based on cell-free vesicle-budding reactions, however, demonstrated that several peroxisomal proteins traffic from the ER to peroxisomes in a COPII vesicle-independent manner (11). These observations point to the existence of vesicular events to mediate the transport of peroxisomal membrane proteins from the ER. In fact, analysis of secretory mutant yeast cells already suggest that part of the ER-associated secretory machinery is involved in peroxisome biogenesis (12).The de novo biogenesis of peroxisomes and the growth/division pathways are usually seen as independent routes; however, these events may be coordinated and, thus, intimately linked. Indeed, peroxisomes need to acquire membrane components to proliferate and it has been proposed that their binding to the cell cortex or to the cytoskeleton allows their partitioning and segregation during cell division (13–15).Among the proteins required for assembly of peroxisomes, the membrane proteins Pex23p and Pex24p play essential roles in the yeast Yarrowia lipolytica (16, 17). Homologs of these two proteins in Saccharomyces cerevisiae are Pex30p, Pex31p, and Pex32p, all containing at least one transmembrane domain and a dysferlin domain as common structural motifs, as well as Pex28p and Pex29p. In S. cerevisiae, these proteins seem to negatively control peroxisomal size and number (18, 19). Interestingly, Pex30p seems to exhibit species-specific differences in the regulation of peroxisome proliferation. While the lack of Pex30p in S. cerevisiae leads to an increase in the number of normal-sized peroxisomes (18), in Pichia pastoris its absence correlates with the appearance of fewer and clustered peroxisomes (20). Although peroxisomes are highly versatile organelles, under given conditions their total number per cell remains fairly constant owing to the delicate balance of proliferation, inheritance and degradation (21, 22). The question is: what are the molecular mechanisms responsible for the spatiotemporal organization of these events?Here, we present data obtained from a dual approach based on quantitative interaction proteomics using stable isotope labeling with amino acids in cell culture (SILAC) (23, 24) and live-cell imaging, revealing for the first time the dynamic interaction network around Pex30p and its function in the organization of ER-to-peroxisome membrane associations. We report the existence of a macromolecular membrane protein complex that acts as a hub for the regulation of peroxisome proliferation and movement. Our data suggest a direct role for the tubular cortical ER and the reticulon homology proteins Rtn1p, Rtn2p, and Yop1p in the regulation of peroxisome biogenesis. Furthermore, as an initially cortical-ER localized protein that interacts with reticulon homology proteins, Pex30p is shown in this work to establish contacts between ER tubules and peroxisomes and to specifically traffic through the ER. In summary, our data reveal a central role for Pex30p in the formation of ER-to-peroxisomes associations that appear to be involved in the coordination of peroxisome biogenesis and maintenance.