The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

How much do disasters cost? A comparison of disaster cost estimates in Australia

Extreme weather events in Australia are common and a large proportion of the population are exposed to such events. Therefore, there is great interest as to how these events impact Australia's society and economy, which requires understanding the current and historical impact of disasters. Despite global efforts to record and cost disaster impacts, no standardised method of collecting and recording data retrospectively yet exists. The lack of standardisation in turn results in a range of different estimates of economic impacts. This paper examines five examples of aggregate disaster loss and impacts of natural disasters in Australia, and comparisons between them reveal significant data shortcomings. The reliability of data sources, and the methodology employed to analyse them can have significant impacts on conclusions regarding the overall cost of disasters, the relative costs of different hazards (disaster types), and the distribution of losses across Australian states. We highlight difficulties with time series comparisons, further complicated by the interdependencies of the databases. We reiterate the need for consistent and comparable data collection and analysis, to respond to the increasing frequency and severity of disasters in Australia

The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase

Folding-competent and folding-defective forms of Ricin A chain have different fates following retrotranslocation from the endoplasmic reticulum

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTAΔ), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER–Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTAΔ, although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate

RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.

Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.This work was supported by NIH Grants RO1-DK099156, RO1-HD060860, and RO1-HL129086 (to K.M.C.); American Heart Association Innovator Award 16IRG27260077 (to K.M.C.); NIH Grant F32-HL134279 (to D.I.M.); American Heart Association Grant 15POST25270006 (to R.B.D.); NIH Grant F31-HL143836 (to N.R.N.); Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/M00015X/2 (to G.L.); and BBSRC Doctoral Training Partnership Grant BB/JO14540/1 (to M.H.)

Changes in somatosensory evoked potentials following an experimental focal ischaemic lesion in thalamus

Experiments have been performed to produce localized thalamic ischaemia in baboons anaesthetised with alpha-chloralose. Somatosensory evoked potentials to median nerve stimulation were recorded in the medial lemniscus. VPL of thalamus and the primary somatosensory cortex. Local blood flow was also recorded by the hydrogen clearance technique in these regions. The early potential recorded in thalamus has been shown to be generated from 3 sources: (i) a positivity generated outside the VPL, (ii) local wavelets, most likely from synaptic activity close to the recording electrode, and (iii) a local overall negativity. The first of these potentials alone remains after thalamic ischaemia. It arises below the level of the thalamus, being very likely generated by the afferent volley in the medial lemniscus, and is seen in the surface-recorded response as the early component P8 (corresponding to P15 in the human).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27315/1/0000336.pd

Hyperglycemia does not affect antigen specific activation and cytolytic killing by CD8+ T cells in vivo

Metabolism is of central importance to T cell survival and differentiation. It is well known that T cells cannot function in the absence of glucose, but it is less clear how they respond to excessive levels of glucose. In this study we investigated how increasing levels of glucose affect T cell-mediated immune responses. We examined the effects of increased levels of glucose on CD8⁺ T cell behaviour in vitro by assessing activation and cytokine production, as well as oxygen consumption rate, extracellular acidification rate and intracellular signalling. In addition, we assessed in vivo proliferation, cytokine production and cytolytic activity of cells in chemically induced diabetic C57BL6 mice. Elevated levels of glucose in in vitro cultures had modest effects on proliferation and cytokine production, while in vivo hyperglycemia had no effect on CD8⁺ T cell proliferation, interferon gamma production or cytolytic killing.This work was supported by the NC3Rs [grant number NC/M001083/1]; the BBSRC [grant number BB/M00015X/2]; the Leverhulme Trust [grant number EM-2015-030]; the Medical Research Council [grant number G0901155]; the Diabetes U.K. [grant number BDA 09/0003840]; the BBSRC-funded Midlands Integrative Biosciences Training Partnership (MIBTP) (K.B.); and the Lollipop Foundation (M.W. as the main grant applicant and A.R. as a co-applicant)

Engineering a Model Cell for Rational Tuning of GPCR Signaling.

G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.BBSR

Parameter identification problems in the modelling of cell motility

We present a novel parameter identification algorithm for the estimation of parameters in models of cell motility using imaging data of migrating cells. Two alternative formulations of the objective functional that measures the difference between the computed and observed data are proposed and the parameter identification problem is formulated as a minimisation problem of nonlinear least squares type. A Levenberg–Marquardt based optimisation method is applied to the solution of the minimisation problem and the details of the implementation are discussed. A number of numerical experiments are presented which illustrate the robustness of the algorithm to parameter identification in the presence of large deformations and noisy data and parameter identification in three dimensional models of cell motility. An application to experimental data is also presented in which we seek to identify parameters in a model for the monopolar growth of fission yeast cells using experimental imaging data. Our numerical tests allow us to compare the method with the two different formulations of the objective functional and we conclude that the results with both objective functionals seem to agree