ESCRT machinery mediates selective microautophagy of endoplasmic reticulum in yeast

ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and the ESCRT machinery as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that the ESCRT machinery directly functions in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis

Herbage growth and utilisation under continuous stocking management

Two field experiments were conducted on a mixed species(Lolium perenne L., Poa annua L., Trifolium repens L.) sward, one in the summer and the other in the autumn of 1979. The objective of the study was to examine the relationship between sward conditions and herbage production. This was done by estimating tissue fluxes in swards maintained as near as possible in a steady state with reference to herbage mass under continuous but variable stocking management with sheep.In the first experiment four separate swards were maintained at herbage mass levels of 500, 700, 1000 and 1700 kg 0M ha ^ for the period May to July inclusive. Tissue flows were estimated from measurements on individually identified grass tillers and clover stolons in the field and determinations of tiller and stolon population densities. Rates of herbage growth and senescence compensated each other in such a way that their resultant, net production, was relatively constant over the range of herbage mass from 800 to 1800 kg OM ha ^. Variations within and between treatments in the utilisation of the three plant species were attributable to the distribution of their foliage within the sward canopy. The herbage intake and intake per bite of the ewes increased with the level of herbage mass maintained, but the time the ewes spent grazing increased,reached a maximum in the 1000 kg OM ha-1 sward, and then declined as the level of maintained herbage mass was increased.In the second experiment, deliberate changes in herbage mass were induced in an attempt to increase the rate of net production above that obtainable in swards maintained at a constant herbage mass.Swards were maintained at either high (1700 kg OM ha HH) or low(700 kg OM ha”\ LL) herbage mass, or manipulated from one state to the other over a period of three weeks in August - September (HL and LH).Rates of growth and senescence compensated each other in such a way that net production was similar in the HH, LL, and LH swards, but inthe HL sward rates of herbage growth, senescence and net production were all reduced.A conceptual model of the sward-animal interface was developed,based upon the results of experiment 1, in which levels of green herbage mass control the rates of herbage growth and senescence,the level of herbage intake and the botanical composition of the diet. A simulation exercise based on this conceptual model was used to examine the implications of basing management decisions on herbage mass or a derivative such as lamina mass. This exercise indicated that lamina was likely to be more useful than herbage mass as the basis for decision making and highlighted the potential of tissue flow analysis as a means of evaluating alternative management systems

Structured articulation of knowledge: The influence of question response structure on recipient attitude

Business today is faced with discontinuity and unpredictable change, which makes many of the structured processes of yesteryear redundant or obsolete. Process-based transactions are being replaced with technology and increasingly organisations are recognising the importance of proactively managing their knowledge transactions, to remain competitive. While research on knowledge sharing is gaining the attention of researchers, almost invariably their focus has been on the factors influencing knowledge transfer at the macro-level in large multi-national organisations. Few have attempted to unravel the complexities of individual-to-individual micro-level knowledge sharing and those that have, for the most part have directed their investigations towards exploring factors that enhance or impede the source individual sharing their knowledge, rather than the recipient's receiving of knowledge. While questioning is implicit in knowledge sharing there are assumptions that underpin the structure of a question and these assumptions affect both the source and the recipient. This study investigates how the structure of a question posed to a source individual when eliciting knowledge, influences the attitude of a recipient individual towards the knowledge they receive from the question response. Drawing upon theoretical assumptions that underpin question structure, three hypotheses are posed to compare binary, open-ended and directed question responses. To test the hypotheses a progression of three independent studies were performed using laboratory and field experiments. The first study conducted in a laboratory, used a contrived scenario case as the knowledge context and the second study replicated this experiment in the field. The last study conducted in a single organisation, used real organisational knowledge as the knowledge context. Recipients of shared knowledge were found to be more favourably disposed towards question responses that were structured in a complex manner; open-ended and directed question responses were more favoured than binary question responses. ii There was no difference in recipient attitude between open-ended and directed question responses and recipient attitude towards the shared knowledge was found to be positively related to their intention to use the knowledge in the future. These findings are of significance as they illustrate the importance of structuring questions in a manner that is consistent with recipients of the shared knowledge being more favourably disposed towards the knowledge they have received. In an environment of ambiguity, complexity and uncertainty where decisions are nonprogrammed, strategic and imperative to the competitiveness of the organisation, no longer is the binary 'Yes' or 'No' compliance or audit style question, with its implicit assumptions, sufficient to elicit knowledge. It is important to recognise that often we do not know what we need to know until it is shared by someone. Further, when shared knowledge is cognitively processed with our current knowledge base, the new knowledge is likely to facilitate more informed decision-making. The more favourably disposed the recipient is towards the knowledge the more likely it is that they will use it in the future; knowledge is transferred

The international political economy of 'actually existing capitalism': Rethinking globalisation and the retreat of the state.

This thesis presents an alternative tradition of classical Marxism capable of understanding what appears to be a shift in power from states to markets over the last two decades. It provides a theory of international political economy which explains both state ownership and control of the economy and its relinquishment, as aspects of 'actually existing capitalism' on a global scale. It is argued that this approach is superior to both Weberian-influenced International Political Economy (IPE), and the current tradition of classical Marxism in International Relations (IR), in that it has the potential to provide a deeper understanding of the apparent 'retreat of the state' as an aspect of so-called 'globalization'. The core contribution of the thesis is a critique of the current classical Marxist approach in International Relations and the proposal of an alternative which differs in its analysis of the space, time and motion of capitalism. It is argued, through a rereading of Capital volumes 1 to 3, that this alternative is truer to Marx's intentions. It is further argued that this more nuanced understanding of capitalism is well-represented through the writings of Hilferding, Bukharin, and Lenin, and is identifiable, though underdeveloped, in the work of contemporary Marxists influenced by these theorists. This alternative tradition of classical Marxism provides an understanding of capitalism in phases of both 'nationalization' and 'privatization', deepening our understanding of capitalism as it 'actually exists'. The thesis has two main tasks. The first is to show that both Weberian-influenced IPE and classical Marxism in IR have an inadequate model of capitalism, a theoretical limitation that has become evident in the globalization debate over 'the retreat of the state'. The second is to suggest an alternative theory of capitalism based on a rereading of Capital volumes 1-3. This theory of 'actually existing capitalism' is better able to capture the complexity of changing state market-relations including what is superficially described as the 'retreat of the state'

The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies

A global corporate census: publicly traded and close companies in 1910

In 1910 the world had almost half a million corporations, only one-hundredth of today's total. About one-fifth—with over half of corporate capital—were publicly tradable, higher portions than today. Most publicly quoted corporations traded in Europe and the British Empire, but most close (private) corporations operated in the US, which, until the 1940s, had more corporations per capita than anywhere else. The 83 countries surveyed here differed markedly in company numbers, corporate capital/GDP ratios, and average corporate size. Enclave economies—dominated by quoted (and often foreign-owned) companies—had the largest average sizes, while other nations had more varied mixes of large quoted corporations and close company small and medium enterprises

Ethanol exposure increases mutation rate through error-prone polymerases

International audienceEthanol is a ubiquitous environmental stressor that is toxic to all lifeforms. Here, we use the model eukaryote Saccharomyces cerevisiae to show that exposure to sublethal ethanol concentrations causes DNA replication stress and an increased mutation rate. Specifically, we find that ethanol slows down replication and affects localization of Mrc1, a conserved protein that helps stabilize the replisome. In addition, ethanol exposure also results in the recruitment of error-prone DNA polymerases to the replication fork. Interestingly, preventing this recruitment through mutagenesis of the PCNA/Pol30 polymerase clamp or deleting specific error-prone polymerases abolishes the mutagenic effect of ethanol. Taken together, this suggests that the mutagenic effect depends on a complex mechanism, where dysfunctional replication forks lead to recruitment of error-prone polymerases. Apart from providing a general mechanistic framework for the mutagenic effect of ethanol, our findings may also provide a route to better understand and prevent ethanol-associated carcinogenesis in higher eukaryotes