The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels

Fast synaptic transmission involves the operation of ionotropic receptors, which are often composed of at least two types of subunit. We have developed a method, based on atomic force microscopy imaging to determine the stoichiometry and subunit arrangement within ionotropic receptors. We showed recently that the P2X(2) receptor for ATP is expressed as a trimer but that the P2X(6) subunit is unable to oligomerize. In this study we addressed the subunit stoichiometry of heteromers containing both P2X(2) and P2X(6) subunits. We transfected tsA 201 cells with both P2X(2) and P2X(6) subunits, bearing different epitope tags. We manipulated the transfection conditions so that either P2X(2) or P2X(6) was the predominant subunit expressed. By atomic force microscopy imaging of isolated receptors decorated with antiepitope antibodies, we demonstrate that when expression of the P2X(2) subunit predominates, the receptors contain primarily 2 x P2X(2) subunits and 1 x P2X(6) subunit. In contrast, when the P2X(6) subunit predominates, the subunit stoichiometry of the receptors is reversed. Our results show that the composition of P2X receptor heteromers is plastic and dependent on the relative subunit expression levels. We suggest that this property of receptor assembly might introduce an additional layer of subtlety into P2X receptor signaling

Analysis of naturally occurring mutations in the human lipodystrophy protein seipin reveals multiple potential pathogenic mechanisms

Peer reviewedPublisher PD

Seipin oligomers can interact directly with AGPAT2 and lipin 1, physically scaffolding critical regulators of adipogenesis

This work was supported by a Merit Scholarship from the Islamic Development Bank (to M.M.U.T.), The Agency for Science, Technology and Research, Singapore (A*STAR) (M.F.M.S), the Medical Research Council (MRC) [NIRG GO800203 and Research Grant MR/L002620/1 (to J.J.R.), Program GrantG09000554 (to S.O.R)], The Wellcome Trust [078986/Z/06/Z (to S.O.R.)], the MRC Centre for Obesity and Related Metabolic Disorders (MRC-CORD) [GO600717] and the NIHR Comprehensive Biomedical Research Centre [CG50826].Peer reviewedPublisher PD

DNA translocation by type III restriction enzymes: a comparison of current models of their operation derived from ensemble and single-molecule measurements

Much insight into the interactions of DNA and enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques—atomic force microscopy (AFM) and magnetic tweezers (MT)—have produced apparently contradictory results when applied to the action of the ATP-dependent type III restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme–enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this ‘Survey and Summary’, we describe several different models put forward for the action of type III restriction enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these enzymes

Study design and protocol for a mixed methods evaluation of an intervention to reduce and break up sitting time in primary school classrooms in the UK: the CLASS PAL (Physically Active Learning) Programme

Introduction: Children engage in a high volume of sitting in school, particularly in the classroom. A number of strategies, such as physically active lessons (termed movement integration (MI)), have been developed to integrate physical activity into this learning environment; however, no single approach is likely to meet the needs of all pupils and teachers. This protocol outlines an implementation study of a primary school-based MI intervention: CLASS PAL (Physically Active Learning) programme. This study aims to (A) determine the degree of implementation of CLASS PAL, (B) identify processes by which teachers and schools implement CLASS PAL and (C) investigate individual (pupil and teacher) level and school-level characteristics associated with implementation of CLASS PAL. Methods and analysis: The intervention will provide teachers with a professional development workshop and a bespoke teaching resources website. The study will use a single group before-and-after design, strengthened by multiple interim measurements. Six state-funded primary schools will be recruited within Leicestershire, UK. Evaluation data will be collected prior to implementation and at four discrete time points during implementation: At measurement 0 (October 2016), school, teacher and pupil characteristics will be collected. At measurements 0 and 3 (June-July 2017), accelerometry, cognitive functioning, self-reported sitting and classroom engagement data will be collected. At measurements 1(December 2016-March 2017) and 3, teacher interviews (also at measurement 4; September-October 2017) and pupil focus groups will be conducted, and at measurements 1 and 2 (April-May 2017), classroom observations. Implementation will be captured through website analytics and ongoing teacher completed logs. Ethics and dissemination: Ethical approval was obtained through the Loughborough University Human Participants Ethics Sub-Committee (Reference number: R16-P115). Findings will be disseminated via practitioner and/or research journals and to relevant regional and national stakeholders through print and online media and dissemination event(s)

Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping

Atomic force microscopy (AFM) allows the study of single protein–DNA interactions such as those observed with the Type I Restriction–Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPγS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein–DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping

A Cell-Free System for Regulated Exocytosis in Pc12 Cells

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca2+ at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca2+-dependent manner dur- ing 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimula- tion. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools