A role for EFR3A during insulin stimulated dispersal of GLUT4 at the plasma membrane

The regulation of blood glucose levels post-prandial relies on insulin-stimulated glucose uptake within adipocytes and striated muscle. The insulin effect in these tissues is a result of the translocation of the facilitative glucose transporter type IV (GLUT4) from intracellular storage vesicles to the plasma membrane. GLUT4 translocation results in the majority of glucose uptake in the body post-prandial. A major contribution to developing Type-2 diabetes is insulin-resistance, which is a result of ineffective insulin signalling, and glucose uptake. GLUT4 is found in insulin sensitive tissue, such as adipose and striated muscle, it is sequestered and stored intracellularly in specialised GLUT4 storage vesicles (GSV) in the absence of insulin. GSVs are a dynamic vesicle which translocate in response to insulin stimulation to the plasma membrane. In the absence of insulin stimulation GSVs which arrive at the PM show reduced fusion to the membrane and are quickly re-endocytosed. Upon insulin stimulation there is an increase in GSVs translocation to the plasma membrane and upon arrival fusion with retention at the plasma membrane is increased. At the plasma membrane GLUT4 arrives within in a cluster. Within this cluster GLUT4 is dynamic but corralled within in the cluster. In the absence of insulin GLUT4 is quickly re-endocytosed in a “kiss-and-run” type event. In the insulin-stimulated cell GLUT4 arrives in clusters which have been observed to disperse from the original site of fusion. In response to insulin stimulation GLUT4 mobility at the plasma membrane is increased with increased dispersal. This indicates that insulin stimulation has an effect on the behaviour of GLUT4 at the plasma membrane. This dispersal is hypothesised to increase GLUT4 dwell time at the plasma membrane increasing the effect of insulin signalling for a greater period of time. The dispersal of GLUT4 at the plasma membrane is an insulin mediated response which has no known molecular mechanism. A genetic screen conducted in S. cerevisiae indicate a role for a mutant allele, fgy1-1, of the protein Efr3. This protein has two homologous mammalian orthologues EFR3A and EFR3B. The mammalian EFR3 is a palymitoylated protein responsible for membrane localisation and as a result the activity of the phosphoinositide kinase, PI4K type IIIα. PI4KIIIα activity is required for generation of phosphoinositide 4-phosphate (PI4P) at the plasma membrane inner leaflet. The phospho-identity of the membrane phosphoinositide has been shown to affect a variety of cellular functions. Mobility of plasma membrane inserted proteins is dictated by the composition of the membrane and cytoskeletal network below the membrane. Single molecule tracking of GLUT4 at the plasma membrane shows increased mobility in the insulin-stimulated cell. Increased mobility and increased dispersal of GLUT4 in response to insulin stimulation has no known molecular mechanism which made EFR3 a promising candidate for further investigation. This thesis aims to investigate EFR3A and of PI4P during insulin stimulated GLUT4 dispersal. The results from these experiments showed that EFR3A is the expressed homolog within adipocyte cell types used in this investigation. Results indicating that in mice with impaired glucose tolerance EFR3A and PI4KIIIα protein levels are increased in insulin responsive striated muscle samples. This indicates a compensatory effect as insulin resistance occurs in these animals. Results showed that increased expression of EFR3A led to increasing the plasma membrane GLUT4 in the absence of insulin stimulation signifying GLUT4 is enriched in the plasma membrane instead of intracellularly within GSVs when EFR3A is over expressed. Inhibition of EFR3A plasma membrane localisation through expression of a cytosolic mutant leads to inhibition of the insulin stimulated increase of GLUT4 at the plasma membrane. This indicates that EFR3A at the plasma membrane is important for insulin stimulated GLUT4 increase. In concurrence with these data inhibition of EFR3A and PI4KIIIα through siRNA depletion resulted in inhibited insulin stimulated glucose uptake. Increased glucose uptake is the end result of insulin stimulation, and inhibition of EFR3A machinery inhibits this activity. Taken together these findings indicate that EFR3A has a positive effect on plasma membrane GLUT4 and insulin stimulated glucose uptake. A role for the generation of PI4P at the plasma membrane is proposed to be behind this positive effect, which future work could aim to elucidate

Recycling of cell surface membrane proteins from yeast endosomes is regulated by ubiquitinated Ist1

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both “early” and “recycling” endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved

SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion

Stakeholder perspectives on contributors to delayed and inaccurate diagnosis of cardiovascular disease and their implications for digital health technologies: a UK-based qualitative study

Objective: The aim of this study is to understand stakeholder experiences of diagnosis of cardiovascular disease (CVD) to support the development of technological solutions that meet current needs. Specifically, we aimed to identify challenges in the process of diagnosing CVD, to identify discrepancies between patient and clinician experiences of CVD diagnosis, and to identify the requirements of future health technology solutions intended to improve CVD diagnosis. Design: Semistructured focus groups and one-to-one interviews to generate qualitative data that were subjected to thematic analysis. Participants: UK-based individuals (N=32) with lived experience of diagnosis of CVD (n=23) and clinicians with experience in diagnosing CVD (n=9). Results: We identified four key themes related to delayed or inaccurate diagnosis of CVD: symptom interpretation, patient characteristics, patient–clinician interactions and systemic challenges. Subthemes from each are discussed in depth. Challenges related to time and communication were greatest for both stakeholder groups; however, there were differences in other areas, for example, patient experiences highlighted difficulties with the psychological aspects of diagnosis and interpreting ambiguous symptoms, while clinicians emphasised the role of individual patient differences and the lack of rapport in contributing to delays or inaccurate diagnosis. Conclusions: Our findings highlight key considerations when developing digital technologies that seek to improve the efficiency and accuracy of diagnosis of CVD

The Rpd3-complex regulates expression of multiple cell surface recycling factors in yeast

Intracellular trafficking pathways control residency and bioactivity of integral membrane proteins at the cell surface. Upon internalisation, surface cargo proteins can be delivered back to the plasma membrane via endosomal recycling pathways. Recycling is thought to be controlled at the metabolic and transcriptional level, but such mechanisms are not fully understood. In yeast, recycling of surface proteins can be triggered by cargo deubiquitination and a series of molecular factors have been implicated in this trafficking. In this study, we follow up on the observation that many subunits of the Rpd3 lysine deacetylase complex are required for recycling. We validate ten Rpd3-complex subunits in recycling using two distinct assays and developed tools to quantify both. Fluorescently labelled Rpd3 localises to the nucleus and complements recycling defects, which we hypothesised were mediated by modulated expression of Rpd3 target gene(s). Bioinformatics implicated 32 candidates that function downstream of Rpd3, which were over-expressed and assessed for capacity to suppress recycling defects of rpd3∆ cells. This effort yielded three hits: Sit4, Dit1 and Ldb7, which were validated with a lipid dye recycling assay. Additionally, the essential phosphatidylinositol-4-kinase Pik1 was shown to have a role in recycling. We propose recycling is governed by Rpd3 at the transcriptional level via multiple downstream target genes

Stakeholder perspectives on contributors to delayed and inaccurate diagnosis of cardiovascular disease and their implications for digital health technologies: a UK-based qualitative study

Objective: The aim of this study is to understand stakeholder experiences of diagnosis of cardiovascular disease (CVD) to support the development of technological solutions that meet current needs. Specifically, we aimed to identify challenges in the process of diagnosing CVD, to identify discrepancies between patient and clinician experiences of CVD diagnosis, and to identify the requirements of future health technology solutions intended to improve CVD diagnosis. Design: Semistructured focus groups and one-to-one interviews to generate qualitative data that were subjected to thematic analysis. Participants: UK-based individuals (N=32) with lived experience of diagnosis of CVD (n=23) and clinicians with experience in diagnosing CVD (n=9). Results: We identified four key themes related to delayed or inaccurate diagnosis of CVD: symptom interpretation, patient characteristics, patient–clinician interactions and systemic challenges. Subthemes from each are discussed in depth. Challenges related to time and communication were greatest for both stakeholder groups; however, there were differences in other areas, for example, patient experiences highlighted difficulties with the psychological aspects of diagnosis and interpreting ambiguous symptoms, while clinicians emphasised the role of individual patient differences and the lack of rapport in contributing to delays or inaccurate diagnosis. Conclusions: Our findings highlight key considerations when developing digital technologies that seek to improve the efficiency and accuracy of diagnosis of CVD

Endosomal cargo recycling mediated by Gpa1 and phosphatidylinositol 3-kinase is inhibited by glucose starvation

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalized protein and lipid cargoes recycle back to the surface efficiently in glucose-replete conditions, but this trafficking is attenuated following glucose starvation. We find that cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for endosome to surface recycling. Furthermore, we find that the yeast Gα subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct Gα subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose that this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and overexpression of Gpa2 alter PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation

Autocrine TNF-α production supports CML stem and progenitor cell survival and enhances their proliferation.

Chronic myeloid leukemia (CML) stem cells are not dependent on BCR-ABL kinase for their survival, suggesting that kinase-independent mechanisms must contribute to their persistence. We observed that CML stem/progenitor cells (SPCs) produce tumor necrosis factor-α (TNF-α) in a kinase-independent fashion and at higher levels relative to their normal counterparts. We therefore investigated the role of TNF-α and found that it supports survival of CML SPCs by promoting nuclear factor κB/p65 pathway activity and expression of the interleukin 3 and granulocyte/macrophage-colony stimulating factor common β-chain receptor. Furthermore, we demonstrate that in CML SPCs, inhibition of autocrine TNF-α signaling via a small-molecule TNF-α inhibitor induces apoptosis. Moreover TNF-α inhibition combined with nilotinib induces significantly more apoptosis relative to either treatment alone and a reduction in the absolute number of primitive quiescent CML stem cells. These results highlight a novel survival mechanism of CML SPCs and suggest a new putative therapeutic target for their eradication.This study was supported by the Glasgow Experimental Cancer Medicine Centre , which is funded by Cancer Research UK and by the Chief Scientist’s Office, Scotland. Cell sorting facilities were funded by the Kay Kendall Leukaemia Fund (KKL501) and the Howat Foundation. Funding was provided by Medical Research Council UK clinical research training fellowship grant G1000288 (P.G.), Cancer Research UK Programme grant C11074/A11008 and the Elimination of Leukaemia Fund (ELF/6/ 29/1) (F.P.), National Institutes of Health, National Cancer Institute research grant R01 CA095684 (R.B.), by the Friends of Paul O’Gorman Leukaemia Research Centre (H.G.J.), and Cancer Research UK Programme grant C11074/A11008 (T.L.H.)