Arp2/3 complex activity in filopodia of spreading cells

Background Cells use filopodia to explore their environment and to form new adhesion contacts for motility and spreading. The Arp2/3 complex has been implicated in lamellipodial actin assembly as a major nucleator of new actin filaments in branched networks. The interplay between filopodial and lamellipodial protrusions is an area of much interest as it is thought to be a key determinant of how cells make motility choices. Results We find that Arp2/3 complex localises to dynamic puncta in filopodia as well as lamellipodia of spreading cells. Arp2/3 complex spots do not appear to depend on local adhesion or on microtubules for their localisation but their inclusion in filopodia or lamellipodia depends on the activity of the small GTPase Rac1. Arp2/3 complex spots in filopodia are capable of incorporating monomeric actin, suggesting the presence of available filament barbed ends for polymerisation. Arp2/3 complex in filopodia co-localises with lamellipodial proteins such as capping protein and cortactin. The dynamics of Arp2/3 complex puncta suggests that they are moving bi-directionally along the length of filopodia and that they may be regions of lamellipodial activity within the filopodia. Conclusion We suggest that filopodia of spreading cells have regions of lamellipodial activity and that this activity affects the morphology and movement of filopodia. Our work has implications for how we understand the interplay between lamellipodia and filopodia and for how actin networks are generated spatially in cells

Phage display selected magnetite interacting Adhirons for shape controlled nanoparticle synthesis

Adhirons are robust, well expressing, peptide display scaffold proteins, developed as an effective alternative to traditional antibody binding proteins for highly specific molecular recognition applications. This paper reports for the first time the use of these versatile proteins for material binding, and as tools for controlling material synthesis on the nanoscale. A phage library of Adhirons, each displaying two variable binding loops, was screened to identify specific proteins able to interact with [100] faces of cubic magnetite nanoparticles. The selected variable regions display a strong preference for basic residues such as lysine. Molecular dynamics simulations of amino acid adsorption onto a [100] magnetite surface provides a rationale for these interactions, with the lowest adsorption energy observed with lysine. These proteins direct the shape of the forming nanoparticles towards a cubic morphology in room temperature magnetite precipitation reactions, in stark contrast to the high temperature, harsh reaction conditions currently used to produce cubic nanoparticles. These effects demonstrate the utility of the selected Adhirons as novel magnetite mineralization control agents using ambient aqueous conditions. The approach we outline with artificial protein scaffolds has the potential to develop into a toolkit of novel additives for wider nanomaterial fabrication

Concentrating Membrane Proteins Using Asymmetric Traps and AC Electric Fields

Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol.2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery2006, 5, 993). However, the structure-function relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structure-function determination of these species (Poo et al. Nature1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a “nested trap” and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins

Taking a hard line with biotemplating: cobalt-doped magnetite magnetic nanoparticle arrays.

Rapid advancements made in technology, and the drive towards miniaturisation, means that we require reliable, sustainable and cost effective methods of manufacturing a wide range of nanomaterials. In this bioinspired study, we take advantage of millions of years of evolution, and adapt a biomineralisation protein for surface patterning of biotemplated magnetic nanoparticles (MNPs). We employ soft-lithographic micro-contact printing to pattern a recombinant version of the biomineralisation protein Mms6 (derived from the magnetotactic bacterium Magnetospirillum magneticum AMB-1). The Mms6 attaches to gold surfaces via a cysteine residue introduced into the N-terminal region. The surface bound protein biotemplates highly uniform MNPs of magnetite onto patterned surfaces during an aqueous mineralisation reaction (with a mean diameter of 90 ± 15 nm). The simple addition of 6% cobalt to the mineralisation reaction maintains the uniformity in grain size (with a mean diameter of 84 ± 14 nm), and results in the production of MNPs with a much higher coercivity (increased from ≈156 Oe to ≈377 Oe). Biotemplating magnetic nanoparticles on patterned surfaces could form a novel, environmentally friendly route for the production of bit-patterned media, potentially the next generation of ultra-high density magnetic data storage devices. This is a simple method to fine-tune the magnetic hardness of the surface biotemplated MNPs, and could easily be adapted to biotemplate a wide range of different nanomaterials on surfaces to create a range of biologically templated devices

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

Plasmon-based determination of macromolecular interactions with membrane-encapsulated nanoparticles

Nanoparticles exhibit various optical properties arising from scattering and absorption due to polariton excitation. The resulting frequency and amplitude is dependent on several factors such as particle size, shape, and dielectric environment. By modifying the environment of the nanoparticle surface, in particular by encapsulating an individual nanoparticle within a membrane bilayer comprising defined phospholipids, these properties may be utilised to interrogate molecular interactions adjacent to the particle surface to useful levels of sensitivity. We describe the underlying rationale of these properties and characterise the preparation and behaviour of the nanoparticles. We indicate the potential this approach may have for sensing and screening in analytical biomolecular technology by demonstrating that it can be utilised to reveal the kinetics of the molecular interactions of membrane associated events. We also indicate that the technique may yield higherorder structural information of the macromolecule-membrane interactions in a highly sensitive manner and discuss the physical origins of these potentially more exotic phenomena

Towards defining the Mechanisms of Alzheimer’s disease based on a contextual analysis of molecular pathways

Alzheimer’s disease (AD) is posing an increasingly profound problem to society. Our genuine understanding of the pathogenesis of AD is inadequate and as a consequence, diagnostic and therapeutic strategies are currently insufficient. The understandable focus of many studies is the identification of molecules with high diagnostic utility however the opportunity to obtain a further understanding of the mechanistic origins of the disease from such putative biomarkers is often overlooked. This study examines the involvement of biomarkers in AD to shed light on potential mechanisms and pathways through which they are implicated in the pathology of this devastating neurodegenerative disorder. The computational tools required to analyse ever-growing datasets in the context of AD are also discussed

Manipulation and sorting of membrane proteins using patterned diffusion-aided ratchets with AC fields in supported lipid bilayers

We present ratchets capable of directing the movement of charged components within supported bilayer lipid membranes. These ratchets make use of asymmetrically patterned features and AC electric fields, and have been demonstrated to transport charged species such as lipids and transmembrane proteins between two reservoirs. Proteins were present in both orientations in the membrane, with only those with their extra-membranous domain orientated away from the glass substrate being mobile. Proteins in the mobile orientation were transported using these ratchets, thereby sorting the two orientations from one another, and creating an area of the membrane containing five times more protein in one orientation than the other