Using size-selected gold clusters on graphene oxide films to aid cryo-transmission electron tomography alignment

A three-dimensional reconstruction of a nano-scale aqueous object can be achieved by taking a series of transmission electron micrographs tilted at different angles in vitreous ice: cryo-Transmission Electron Tomography. Presented here is a novel method of fine alignment for the tilt series. Size-selected gold clusters of ~2.7 nm (Au(561 ± 14)), ~3.2 nm (Au(923 ± 22)), and ~4.3 nm (Au(2057 ± 45)) in diameter were deposited onto separate graphene oxide films overlaying holes on amorphous carbon grids. After plunge freezing and subsequent transfer to cryo-Transmission Electron Tomography, the resulting tomograms have excellent (de-)focus and alignment properties during automatic acquisition. Fine alignment is accurate when the evenly distributed 3.2 nm gold particles are used as fiducial markers, demonstrated with a reconstruction of a tobacco mosaic virus. Using a graphene oxide film means the fiducial markers are not interfering with the ice bound sample and that automated collection is consistent. The use of pre-deposited size-selected clusters means there is no aggregation and a user defined concentration. The size-selected clusters are mono-dispersed and can be produced in a wide size range including 2–5 nm in diameter. The use of size-selected clusters on a graphene oxide films represents a significant technical advance for 3D cryo-electron microscopy

Early Signaling in Primary T Cells Activated by Antigen Presenting Cells Is Associated with a Deep and Transient Lamellal Actin Network

Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches

Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis.

Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1-3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4-8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome.We thank the Wellcome Trust for funding to GMG (075880) and CIMR (100140).This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.cub.2015.10.02

Quantitative silencing of EGFP reporter gene by self-assembled siRNA lipoplexes of LinOS and cholesterol

[Image: see text] Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N(4),N(9)-diacyl spermine conjugate, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N(4),N(9)-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106–118 nm, compared to 194–356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1:2 lipoplexes shows the formation of multilamellar spheres with a size of ∼100 nm, in good agreement with the particle size measured by DLS. The constant distance between lamellar repeats is ∼6 nm, with the electron-dense layers fitting a monolayer of siRNA. AlamarBlue cell viability assay showed that the lipoplexes resulted in cell viability ≥81%, with LinOS/Chol 1:2 mixtures resulting in cell viabilities of 89% and 94% at siRNA 15 nM and 3.75 nM respectively. These results show that lipoplexes of siRNA and LinOS/Chol mixtures prepared by the direct mixing of the lipid mixture and siRNA, without any preceding preformulation steps, result in enhanced siRNA delivery and EGFP knockdown, with excellent cell viability. Thus, LinOS/Chol 1:2 mixture is a promising candidate as a nontoxic nonviral siRNA vector

Social crisis: reflection on the economic, social and educational alternatives from a gender perspective

Colección ReSed. Es un artículo de ReSed Nº 3, perteneciente al monográfico Crisis Social, Educación y Desarrollo Profesional. Coordinación del Monográfico: Dra. Montserrat Vargas Vergara Dirección de ReSed: Dra. A-Beatriz Pérez-GonzálezEn el presente artículo hacemos una reflexión sobre cómo la actual crisis financiera plantea una crisis de valores, que nos lleva a la necesidad de reorganización en lo económico, social y educativo. La interpretación del problema la hacemos desde un posicionamiento de estudio de género, donde veremos cómo el feminismo traza alternativas viables. Las perspectivas de la economía feminista o economía del bien común son algunas de las alternativas que vamos a reflejar, al mismo tiempo que describimos una realidad condicionada por el género, no siempre asumida por los gobernantes. La propuesta que surge de esta reflexión está basada en el cuidado equilibrado de todos los miembros de la sociedad, promoviendo una educación igualitaria y más libre entre los géneros; nuevos caminos que se hacen necesarios para construir una sociedad más equitativa y sostenible y que favorezcan el desarrollo personal, social y laboral de todos sus miembros

A de novo virus-like topology for synthetic virions

A de novo ultra-small topology of viral assembly is reported. The design is a tri-faceted coiled-coil peptide helix, which self-assembles into monodisperse, anionic virions able to encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, the virions share the same physical characteristics of viruses being anionic, non-aggregating, abundant, hollow and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virions reported to date with the ability to adapt and transfer small and large nucleic acids thus offering a promising solution for engineering bespoke artificial viruses with desired functions

Infectious bronchitis virus nonstructural protein 4 alone induces membrane pairing

Positive-strand RNA viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral RNA synthesis. Infectious bronchitis virus (IBV), a pathogenic avian of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (DMVs), zippered endoplasmic reticulum (zER) and tethered vesicles, known as spherules. These membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. In this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of IBV were compared. Three non-structural transmembrane proteins, nsp3, nsp4, and nsp6, were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. In contrast to previously studied coronaviruses, IBV nsp4 alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate DMVs. This indicates that although nsp4 is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble IBV replicative structures. This study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family

A general role for TANGO1, encoded by MIA3, in secretory pathway organization and function

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum (ER), which is an essential process in eukaryotic cells. In vertebrates, the MIA3 gene encodes two major forms of transport and Golgi organization protein 1 (TANGO1S and TANGO1L), which have previously been implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics and proteomics, we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function, including having limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 (also known as LMAN1) and SURF4 to the ER–Golgi intermediate compartment and dramatic changes to the ultrastructure of the ER–Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo, including small soluble proteins. Our data support a general role for MIA3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells