A journey through the exocytic pathway

International audienc

Force measurements in E-cadherin–mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42

We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell–cell adhesion. The force required to separate E-cadherin–expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin–expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin–based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces

A Machine Learning Based Body Force Model for Analysis of Fan-Airframe Aerodynamic Interactions

Body force modeling is a numerical strategy that allows an accurate representation of the aerodynamics of turbomachinery blade rows at a reduced computational cost, making it suitable for predicting fan-airframe aerodynamic interactions in boundary layer ingestion (BLI) propulsive architectures. This paper focuses on a new approach for building the body force representation using a machine learning technique, rather than analytically modeling the effects of the blades in the flow. This methodology is developed and assessed in a distorted inflow case representative of a BLI configuration and compared to a full annulus unsteady computation

Crystal structure of Sar1-GDP at 1.7 Å resolution and the role of the NH2 terminus in ER export

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the ER. We report the 1.7-Å structure of Sar1 and find that consistent with the sequence divergence of Sar1 from Arf family GTPases, Sar1 is structurally distinct. In particular, we show that the Sar1 NH2 terminus contains two regions: an NH2-terminal extension containing an evolutionary conserved hydrophobic motif that facilitates membrane recruitment and activation by the mammalian Sec12 guanine nucleotide exchange factor, and an α1' amphipathic helix that contributes to interaction with the Sec23/24 complex that is responsible for cargo selection during ER export. We propose that the hydrophobic Sar1 NH2-terminal activation/recruitment motif, in conjunction with the α1' helix, mediates the initial steps in COPII coat assembly for export from the ER

BEST1 Gene Therapy Corrects a Diffuse Retina-Wide Microdetachment Modulated by Light Exposure

Mutations in the BEST1 gene cause detachment of the retina and degeneration of photoreceptor (PR) cells due to a primary channelopathy in the neighboring retinal pigment epithelium (RPE) cells. The pathophysiology of the interaction between RPE and PR cells preceding the formation of retinal detachment remains not well-understood. Our studies of molecular pathology in the canine BEST1 disease model revealed retina-wide abnormalities at the RPE-PR interface associated with defects in the RPE microvillar ensheathment and a cone PR-associated insoluble interphotoreceptor matrix. In vivo imaging demonstrated a retina-wide RPE-PR microdetachment, which contracted with dark adaptation and expanded upon exposure to a moderate intensity of light. Subretinal BEST1 gene augmentation therapy using adeno-associated virus 2 reversed not only clinically detectable subretinal lesions but also the diffuse microdetachments. Immunohistochemical analyses showed correction of the structural alterations at the RPE-PR interface in areas with BEST1 transgene expression. Successful treatment effects were demonstrated in three different canine BEST1 genotypes with vector titers in the 0.1-to-5E11 vector genomes per mL range. Patients with biallelic BEST1 mutations exhibited large regions of retinal lamination defects, severe PR sensitivity loss, and slowing of the retinoid cycle. Human translation of canine BEST1 gene therapy success in reversal of macro- and microdetachments through restoration of cytoarchitecture at the RPE-PR interface has promise to result in improved visual function and prevent disease progression in patients affected with bestrophinopathies

Differential roles of CCL2 and CCR2 in host defense to coronavirus infection.

The CC chemokine ligand 2 (CCL2, monocyte chemoattractant protein-1) is important in coordinating the immune response following microbial infection by regulating T cell polarization as well as leukocyte migration and accumulation within infected tissues. The present study examines the consequences of mouse hepatitis virus (MHV) infection in mice lacking CCL2 (CCL2(-/-)) in order to determine if signaling by this chemokine is relevant in host defense. Intracerebral infection of CCL2(-/-) mice with MHV did not result in increased morbidity or mortality as compared to either wild type or CCR2(-/-) mice and CCL2(-/-) mice cleared replicating virus from the brain. In contrast, CCR2(-/-) mice displayed an impaired ability to clear virus from the brain that was accompanied by a reduction in the numbers of antigen-specific T cells as compared to both CCL2(-/-) and wild-type mice. The paucity in T cell accumulation within the central nervous system (CNS) of MHV-infected CCR2(-/-) mice was not the result of either a deficiency in antigen-presenting cell (APC) accumulation within draining cervical lymph nodes (CLN) or the generation of virus-specific T cells within this compartment. A similar reduction in macrophage infiltration into the CNS was observed in both CCL2(-/-) and CCR2(-/-) mice when compared to wild-type mice, indicating that both CCL2 and CC chemokine receptor 2 (CCR2) contribute to macrophage migration and accumulation within the CNS following MHV infection. Together, these data demonstrate that CCR2, but not CCL2, is important in host defense following viral infection of the CNS, and CCR2 ligand(s), other than CCL2, participates in generating a protective response

A broadband x-ray study of the Geminga pulsar with NuSTAR and XMM-Newton

We report on the first hard X-ray detection of the Geminga pulsar above 10 keV using a 150 ks observation with the NuSTAR observatory. The double-peaked pulse profile of non-thermal emission seen in the soft X-ray band persists at higher energies. Broadband phase-integrated spectra over the 0.2-20 keV band with NuSTAR and archival XMM-Newton data do not fit to a conventional two-component model of a blackbody plus power-law, but instead exhibit spectral hardening above ~5 keV. We find two spectral models fit the data well: (1) a blackbody (kT1 ~ 42 eV) with a broken power-law (Gamma1 ~ 2.0, Gamma2 ~ 1.4 and Ebreak ~ 3.4 keV), and (2) two blackbody components (kT1 ~ 44 eV and kT2 ~ 195 eV) with a power-law component (Gamma ~ 1.7). In both cases, the extrapolation of the Rayleigh-Jeans tail of the thermal component is consistent with the UV data, while the non-thermal component overpredicts the near-infrared data, requiring a spectral flattening at E ~ 0.01 - 1 keV. While strong phase variation of the power-law index is present below ~5 keV, our phase-resolved spectroscopy with NuSTAR indicates that another hard non-thermal component with Gamma ~ 1.3 emerges above ~5 keV. The spectral hardening in non-thermal X-ray emission as well as spectral flattening between the optical and X-ray bands argue against the conjecture that a single power-law may account for multi-wavelength non-thermal spectra of middle-aged pulsars.Comment: Accepted to Ap

NuSTAR observations of magnetar 1e 1841-045

We report new spectral and temporal observations of the magnetar 1E 1841-045 in the Kes 73 supernova remnant obtained with the Nuclear Spectroscopic Telescope Array. Combined with new Swift and archival XMM-Newton and Chandra observations, the phase-averaged spectrum is well characterized by a blackbody plus double power law, in agreement with previous multimission X-ray results. However, we are unable to reproduce the spectral results reported based on Suzaku observations. The pulsed fraction of the source is found to increase with photon energy. The measured rms pulsed fractions are similar to 12% and similar to 17% at similar to 20 and similar to 50 keV, respectively. We detect a new feature in the 24-35 keV band pulse profile that is uniquely double peaked. This feature may be associated with a possible absorption or emission feature in the phase-resolved spectrum. We fit the X-ray data using the recently developed electron-positron outflow model by Beloborodov for the hard X-ray emission from magnetars. This produces a satisfactory fit, allowing a constraint on the angle between the rotation and magnetic axes of the neutron star of similar to 20 degrees and on the angle between the rotation axis and line of sight of similar to 50 degrees. In this model, the soft X-ray component is inconsistent with a single blackbody; adding a second blackbody or a power-law component fits the data. The two-blackbody interpretation suggests a hot spot of temperature kT approximate to 0.9 keV occupying similar to 1% of the stellar surface