Direct measurement of Gag–Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy

During retrovirus assembly, the polyprotein Gag directs protein multimerization, membrane binding, and RNA packaging. It is unknown whether assembly initiates through Gag–Gag interactions in the cytosol or at the plasma membrane. We used two fluorescence techniques—two-photon fluorescence resonance energy transfer and fluorescence correlation spectroscopy—to examine Rous sarcoma virus Gag–Gag and –membrane interactions in living cells. Both techniques provide strong evidence for interactions between Gag proteins in the cytoplasm. Fluorescence correlation spectroscopy measurements of mobility suggest that Gag is present in large cytosolic complexes, but these complexes are not entirely composed of Gag. Deletion of the nucleocapsid domain abolishes Gag interactions and membrane targeting. Deletion of the membrane-binding domain leads to enhanced cytosolic interactions. These results indicate that Gag–Gag interactions occur in the cytosol, are mediated by nucleocapsid domain, and are necessary for membrane targeting and budding. These methods also have general applicability to in vivo studies of protein–protein and –membrane interactions involved in the formation of complex macromolecular structures

Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcɛRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcɛRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcɛRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcɛRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcɛRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein–protein interactions in intact, living cells

and Stable Core-Shell Fluorescence Silica Nanoparticles

ABSTRACT A class of highly fluorescent and photostable core−shell nanoparticles from a modified Sto 1ber synthesis in the size range of 20−30 nm is described. These nanoparticles are monodisperse in solution, 20 times brighter, and more photostable than their constituent fluorophore, and are amenable to specific labeling of biological macromolecules for bioimaging experiments. The photophysical characteristics of the encapsulated fluorophores differ from their solution properties. This raises the possibility of tuning nanoparticle structure toward enhanced radiative properties, making them an attractive material platform for a diverse range of applications. Fluorescent nanoparticles have tremendous promise as indicators and photon sources for a number of biotechnological and information technology applications such as biological imaging, sensor technology, microarrays, and optical computing. 1 These applications all require size-controlled, monodisperse, bright nanoparticles that can be specifically conjugated to biological macromolecules or arranged in higherorder structures. Nanoparticles with core-shell architecture have the added benefit of providing a robust platform for incorporating diverse functionalities into a single nanoparticle. 2 We have developed a novel class of multifunctional silicabased fluorescent nanoparticles using a synthesis based on the Stöber method. The Stöber synthesis of colloidal silica, by which monodisperse nano-to micrometer sized silica particles may be obtained, was first described in 1968. 3 Van Blaaderen and co-workers first reported the covalent incorporation of organic fluorophores into Stöber colloidal silica and the synthesis of fluorescent silica nanoparticles in the hundreds of nanometers size range. Fluorescence correlation spectroscopy (FCS) is used as a primary investigative tool to characterize these nanoparticles. FCS has now become a common tool for characterizing the properties of fluorescent moieties in solution

New parton distributions in fixed flavour factorization scheme from recent deep-inelastic-scattering data

We present our QCD analysis of the proton structure function $F_2^p(x,Q^2)$ to determine the parton distributions at the next-to-leading order (NLO). The heavy quark contributions to $F_2^i(x,Q^2)$, with $i$ = $c$, $b$ have been included in the framework of the `fixed flavour number scheme' (FFNS). The results obtained in the FFNS are compared with available results such as the general-mass variable-flavour-number scheme (GM-VFNS) and other prescriptions used in global fits of PDFs. In the present QCD analysis, we use a wide range of the inclusive neutral-current deep-inelastic-scattering (NC DIS) data, including the most recent data for charm $F_2^c$, bottom $F_2^b$, longitudinal $F_L$ structure functions and also the reduced DIS cross sections $\sigma_{r,NC}^\pm$ from HERA experiments. The most recent HERMES data for proton and deuteron structure functions are also added. We take into account ZEUS neutral current $e^ \pm p$ DIS inclusive jet cross section data from HERA together with the recent Tevatron Run-II inclusive jet cross section data from CDF and D{\O}. The impact of these recent DIS data on the PDFs extracted from the global fits are studied. We present two families of PDFs, {\tt KKT12} and {\tt KKT12C}, without and with HERA `combined' data sets on $e^{\pm}p$ DIS. We find these are in good agreement with the available theoretical models.Comment: 23 pages, 26 figures and 4 tables. V3: Only few comments and references added in the replaced version, results unchanged. Code can be found at http://particles.ipm.ir/links/QCD.ht

Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates

<p>Abstract</p> <p>Background</p> <p>Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).</p> <p>Results</p> <p>Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species.</p> <p>Conclusions</p> <p>We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.</p

(Re)Moralizing the suicide debate

Contemporary approaches to the study of suicide tend to examine suicide as a medical or public health problem rather than a moral problem, avoiding the kinds of judgements that have historically characterised discussions of the phenomenon. But morality entails more than judgement about action or behaviour, and our understanding of suicide can be enhanced by attending to its cultural, social, and linguistic connotations. In this work, I offer a theoretical reconstruction of suicide as a form of moral experience that delineates five distinct, yet interrelated domains of understanding – the temporal, the relational, the existential, the ontological, and the linguistic. Attention to each of these domains, I argue, not only enriches our understanding of the moral realm, but provides a heuristic for examining the moral traditions and practices which constitute contemporary understandings of suicide. Keywords: Suicide; philosophy; social values; humanitie

Perceptual Learning in the Absence of Task or Stimulus Specificity

Performance on most sensory tasks improves with practice. When making particularly challenging sensory judgments, perceptual improvements in performance are tightly coupled to the trained task and stimulus configuration. The form of this specificity is believed to provide a strong indication of which neurons are solving the task or encoding the learned stimulus. Here we systematically decouple task- and stimulus-mediated components of trained improvements in perceptual performance and show that neither provides an adequate description of the learning process. Twenty-four human subjects trained on a unique combination of task (three-element alignment or bisection) and stimulus configuration (vertical or horizontal orientation). Before and after training, we measured subjects' performance on all four task-configuration combinations. What we demonstrate for the first time is that learning does actually transfer across both task and configuration provided there is a common spatial axis to the judgment. The critical factor underlying the transfer of learning effects is not the task or stimulus arrangements themselves, but rather the recruitment of commons sets of neurons most informative for making each perceptual judgment