Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

AbstractClathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.</jats:p

A saposin-lipoprotein nanoparticle system for membrane proteins.

A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state

A smart and responsive crystalline porous organic cage membrane with switchable pore apertures for graded molecular sieving

Membranes with high selectivity offer an attractive route to molecular separations, where technologies such as distillation and chromatography are energy intensive. However, it remains challenging to fine tune the structure and porosity in membranes, particularly to separate molecules of similar size. Here, we report a process for producing composite membranes that comprise crystalline porous organic cage films fabricated by interfacial synthesis on a polyacrylonitrile support. These membranes exhibit ultrafast solvent permeance and high rejection of organic dyes with molecular weights over 600 g mol-1. The crystalline cage film is dynamic, and its pore aperture can be switched in methanol to generate larger pores that provide increased methanol permeance and higher molecular weight cut-offs (1,400 g mol-1). By varying the water/methanol ratio, the film can be switched between two phases that have different selectivities, such that a single, 'smart' crystalline membrane can perform graded molecular sieving. We exemplify this by separating three organic dyes in a single-stage, single-membrane process

A solution-processable dissymmetric porous organic cage

A dissymmetric, soluble, porous organic cage from a low-cost racemic precursor.</p

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers.

Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles

Spatial Epidemiology: Current Approaches and Future Challenges

Spatial epidemiology is the description and analysis of geographic variations in disease with respect to demographic, environmental, behavioral, socioeconomic, genetic, and infectious risk factors. We focus on small-area analyses, encompassing disease mapping, geographic correlation studies, disease clusters, and clustering. Advances in geographic information systems, statistical methodology, and availability of high-resolution, geographically referenced health and environmental quality data have created unprecedented new opportunities to investigate environmental and other factors in explaining local geographic variations in disease. They also present new challenges. Problems include the large random component that may predominate disease rates across small areas. Though this can be dealt with appropriately using Bayesian statistics to provide smooth estimates of disease risks, sensitivity to detect areas at high risk is limited when expected numbers of cases are small. Potential biases and confounding, particularly due to socioeconomic factors, and a detailed understanding of data quality are important. Data errors can result in large apparent disease excess in a locality. Disease cluster reports often arise nonsystematically because of media, physician, or public concern. One ready means of investigating such concerns is the replication of analyses in different areas based on routine data, as is done in the United Kingdom through the Small Area Health Statistics Unit (and increasingly in other European countries, e.g., through the European Health and Environment Information System collaboration). In the future, developments in exposure modeling and mapping, enhanced study designs, and new methods of surveillance of large health databases promise to improve our ability to understand the complex relationships of environment to health

Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1

Do residents’ perceptions of being well-placed and objective presence of local amenities match? A case study in West Central Scotland, UK

Background:&lt;p&gt;&lt;/p&gt; Recently there has been growing interest in how neighbourhood features, such as the provision of local facilities and amenities, influence residents’ health and well-being. Prior research has measured amenity provision through subjective measures (surveying residents’ perceptions) or objective (GIS mapping of distance) methods. The latter may provide a more accurate measure of physical access, but residents may not use local amenities if they do not perceive them as ‘local’. We believe both subjective and objective measures should be explored, and use West Central Scotland data to investigate correspondence between residents’ subjective assessments of how well-placed they are for everyday amenities (food stores, primary and secondary schools, libraries, pharmacies, public recreation), and objective GIS-modelled measures, and examine correspondence by various sub-groups.&lt;p&gt;&lt;/p&gt; Methods:&lt;p&gt;&lt;/p&gt; ArcMap was used to map the postal locations of ‘Transport, Health and Well-being 2010 Study’ respondents (n = 1760), and the six amenities, and the presence/absence of each of them within various straight-line and network buffers around respondents’ homes was recorded. SPSS was used to investigate whether objective presence of an amenity within a specified buffer was perceived by a respondent as being well-placed for that amenity. Kappa statistics were used to test agreement between measures for all respondents, and by sex, age, social class, area deprivation, car ownership, dog ownership, walking in the local area, and years lived in current home.&lt;p&gt;&lt;/p&gt; Results:&lt;p&gt;&lt;/p&gt; In general, there was poor agreement (Kappa &lt;0.20) between perceptions of being well-placed for each facility and objective presence, within 800 m and 1000 m straight-line and network buffers, with the exception of pharmacies (at 1000 m straight-line) (Kappa: 0.21). Results varied between respondent sub-groups, with some showing better agreement than others. Amongst sub-groups, at 800 m straight-line buffers, the highest correspondence between subjective and objective measures was for pharmacies and primary schools, and at 1000 m, for pharmacies, primary schools and libraries. For road network buffers under 1000 m, agreement was generally poor.&lt;p&gt;&lt;/p&gt; Conclusion:&lt;p&gt;&lt;/p&gt; Respondents did not necessarily regard themselves as well-placed for specific amenities when these amenities were present within specified boundaries around their homes, with some exceptions; the picture is not clear-cut with varying findings between different amenities, buffers, and sub-groups

Transmembrane helix dynamics of bacterial chemoreceptors supports a piston model of signalling.

Transmembrane α-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock α-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n = 100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors

Central nervous system and muscular bundles preserved in a 240 million year old giant bristletail (Archaeognatha: Machilidae)

Among the incomparably diverse group of insects no cases of central nervous system (CNS) preservation have been so far described in compression fossils. A third of the fossil insects collected from a 240-239 million year old (Ma) level at Monte San Giorgio UNESCO World Heritage (SwitzerlandItaly) underwent phosphatization, resulting in the extraordinary preservation of soft tissues. Here we describe Gigamachilis triassicus gen. et sp. nov. (Archaeognatha: Machiloidea: Machilidae) that, with an estimated total length of similar to 80 millimeters, represents the largest apterygote insect ever recorded. The holotype preserves: (i) components of the CNS represented by four abdominal ganglia, optic lobes with neuropils and compound retina;(ii) muscular bundles. Moreover, G. triassicus, possessing morphological features that prompt its assignment to the extant archaeognathan ingroup Machilidae, places the origin of modern lineages to Middle Triassic. Interestingly, at Monte San Giorgio, in the same stratigraphic unit the modern morphology of G. triassicus co-occurs with the ancient one represented by Dasyleptus triassicus (Archaeognatha: dagger Monura). Comparing these two types of body organization we provide a new reconstruction of the possible character evolution leading towards modern archaeognathan forms, suggesting the acquisition of novel features in a lineage of apterygote insects during the Permian or the Lower Triassic