Isolation of human mitotic protein phosphatase complexes: identification of a complex between protein phosphatase 1 and the RNA helicase Ddx21

Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation. The phosphoprotein phosphatases PP1 and PP2A are paramount for the timely execution of mitotic entry and exit but their interaction partners and substrates are still largely unresolved. High throughput, mass-spectrometry based studies have limited sensitivity for the detection of low-abundance and transient complexes, a typical feature of many protein phosphatase complexes. Moreover, the limited timeframe during which mitosis takes place reduces the likelihood of identifying mitotic phosphatase complexes in asynchronous cells. Hence, numerous mitotic protein phosphatase complexes still await identification. Here we present a strategy to enrich and identify serine/threonine protein phosphatase complexes at the mitotic spindle. We thus identified a nucleolar RNA helicase, Ddx21/Gu, as a novel, direct PP1 interactor. Furthermore, our results place PP1 within the toposome, a Topoisomerase II alpha (TOPOIIα) containing complex with a key role in mitotic chromatin regulation and cell cycle progression, possibly via regulated protein phosphorylation. This study provides a strategy for the identification of further mitotic PP1 partners and the unravelling of PP1 functions during mitosis.     

Alternative splicing of a multi-drug transporter from Pseudoperonospora cubensis generates an RXLR effector protein that elicits a rapid cell death

Pseudoperonospora cubensis, an obligate oomycete pathogen, is the causal agent of cucurbit downy mildew, a foliar disease of global economic importance. Similar to other oomycete plant pathogens, Ps. cubensis has a suite of RXLR and RXLR-like effector proteins, which likely function as virulence or avirulence determinants during the course of host infection. Using in silico analyses, we identified 271 candidate effector proteins within the Ps. cubensis genome with variable RXLR motifs. In extending this analysis, we present the functional characterization of one Ps. cubensis effector protein, RXLR protein 1 (PscRXLR1), and its closest Phytophthora infestans ortholog, PITG_17484, a member of the Drug/Metabolite Transporter (DMT) superfamily. To assess if such effector-non-effector pairs are common among oomycete plant pathogens, we examined the relationship(s) among putative ortholog pairs in Ps. cubensis and P. infestans. Of 271 predicted Ps. cubensis effector proteins, only 109 (41%) had a putative ortholog in P. infestans and evolutionary rate analysis of these orthologs shows that they are evolving significantly faster than most other genes. We found that PscRXLR1 was up-regulated during the early stages of infection of plants, and, moreover, that heterologous expression of PscRXLR1 in Nicotiana benthamiana elicits a rapid necrosis. More interestingly, we also demonstrate that PscRXLR1 arises as a product of alternative splicing, making this the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene to a functional effector protein. Taken together, these data suggest a role for PscRXLR1 in pathogenicity, and, in total, our data provide a basis for comparative analysis of candidate effector proteins and their non-effector orthologs as a means of understanding function and evolutionary history of pathogen effectors.     

Stressful environments can indirectly select for increased longevity

Longevity is modulated by a range of conserved genes in eukaryotes, but it is unclear how variation in these genes contributes to the evolution of longevity in nature. Mutations that increase life span in model organisms typically induce trade‐offs which lead to a net reduction in fitness, suggesting that such mutations are unlikely to become established in natural populations. However, the fitness consequences of manipulating longevity have rarely been assessed in heterogeneous environments, in which stressful conditions are encountered. Using laboratory selection experiments, we demonstrate that long‐lived, stress‐resistant Caenorhabditis elegans age‐1(hx546) mutants have higher fitness than the wild‐type genotype if mixed genotype populations are periodically exposed to high temperatures when food is not limited. We further establish, using stochastic population projection models, that the age‐1(hx546) mutant allele can confer a selective advantage if temperature stress is encountered when food availability also varies over time. Our results indicate that heterogeneity in environmental stress may lead to altered allele frequencies over ecological timescales and indirectly drive the evolution of longevity. This has important implications for understanding the evolution of life‐history strategies.     

Analysis of Genome Sequences from Plant Pathogenic Rhodococcus Reveals Genetic Novelties in Virulence Loci

Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.     

Failed gene conversion leads to extensive end processing and chromosomal rearrangements in fission yeast

Loss of heterozygosity (LOH), a causal event in cancer and human genetic diseases, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms by which such extensive LOH arises, and how it is suppressed in normal cells is poorly understood. We have developed a genetic system to investigate the mechanisms of DNA double‐strand break (DSB)‐induced extensive LOH, and its suppression, using a non‐essential minichromosome, Ch¹⁶, in fission yeast. We find extensive LOH to arise from a new break‐induced mechanism of isochromosome formation. Our data support a model in which Rqh1 and Exo1‐dependent end processing from an unrepaired DSB leads to removal of the broken chromosome arm and to break‐induced replication of the intact arm from the centromere, a considerable distance from the initial lesion. This process also promotes genome‐wide copy number variation. A genetic screen revealed Rhp51, Rhp55, Rhp57 and the MRN complex to suppress both isochromosome formation and chromosome loss, in accordance with these events resulting from extensive end processing associated with failed homologous recombination repair.     

THE FOOD OF RED GROUSE CHICKS LAGOPUS L. SCOTICUS

The diet of young Red Grouse was studied from the crop contents of 211 chicks caught on a moor in northeast Scotland. The food was more varied than that of adults, but shoot tips of heather usually formed at least 75% (by dry weight) of their diet, as in adults. Arthropods, particularly Diptera, were taken frequently by chicks up to three weeks old, but they formed less than 5% (by dry weight) of the diet then. The chicks ate more invertebrates when more were available. There was no evidence that chick survival was related to the abundance of invertebrates. The chicks ate heather containing more nitrogen and phosphorus than that eaten by adults at the same time of year.     

Receptor‐mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy

Autophagosome formation requires multiple autophagy‐related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome‐wide CRISPR screens identifying genes required for NBR1 flux in ATG7^KO cells. We find that ATG7‐independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1''s ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin‐independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor‐mediated clustering of upstream autophagy factors drives continued autophagosome formation.     

Utilisation of different ages of heather on three Scottish moors by red grouse, mountain hares, sheep and red deer

Utilisation of heather at three Scottish moors, by red grouse, mountain hares, sheep and red deer, was measured by collecting their faecal droppings regularly from plots on heather patches of different ages. Rates of heather growth differed between moors, and preferences of the four species, all grazers of heather, were related more closely to its height than to its age. Thus hares spent most time on ground with heather less than 15 cm high, sheep less than 20 cm, grouse preferred heather 10–30 cm high and deer spent most time on heather over 25 cm high.
Differences between outer, middle and inner plots on patches at one of the moors in dicated that grouse are reluctant to move far from cover into areas of newly burned ground, and reluctant to move far into patches of tall, dense heather, when these are next to shorter material. Hares preferred the middle of patches on newly burned ground and very young heather.
At two of the moors, but not the third, there was evidence that heavy grazing by hares in winter reduced the amount of young heather available to hares and grouse in spring. This potential reduction in the quality of the spring diet of grouse is discussed In relation to previously reported associations between numbers of hares and grouse at those two moors.