Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated ...analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate-based inhibitors inside the cell. Because of the existence of metabolic feedback loops, they also act to prevent the de novo synthesis of the natural substrates, resulting in a global, family-wide shutdown of sialyl- and/or fucosyltransferases and remodeling of cell-surface glycans. As an example of the functional consequences, the inhibitors substantially reduce expression of the sialylated and fucosylated ligand sialyl Lewis X on myeloid cells, resulting in loss of selectin binding and impaired leukocyte rolling.
Increased ligand binding to cellular integrins (“activation”) plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis, hemostasis, and ...thrombosis 1–5. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering 6 and depends on integrin-talin interactions 5. Loss of kindlins results in reduced activation of integrins 7–13. Kindlins might promote talin binding to integrins through a cooperative mechanism 5, 14–16; however, kindlins do not increase talin association with integrins 17. Here, we report that, unlike talin head domain (THD), kindlin-3 has little effect on the affinity of purified monomeric αIIbβ3, and it does not enhance activation by THD. Furthermore, studies with ligands of varying valency show that kindlins primarily increase cellular αIIbβ3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduces αIIbβ3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduces the clustering of ligand-occupied αIIbβ3 as revealed by total internal reflection fluorescence and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbβ3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.
•Kindlins cluster αIIbβ3 integrins to increase multivalent ligand binding•Kindlins have little direct effect on αIIbβ3 integrin monomer affinity•These data reveal how kindlin and talin cooperate to activate integrins
CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody ...antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177pos and CD177neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration.
•Ligation of CD177, a GPI-linked surface protein expressed selectively on neutrophils, blocks neutrophil migration independently of PECAM-1.•Blockade reflects activation through β2 integrins, immobilizing cells via stronger integrin attachments and impaired chemokine signaling.
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Lymphocyte function-associated antigen-1 (LFA-1) is a heterodimeric integrin consisting of α(L) (gene name, Itgal) and β(2) (gene name, Itgb2) subunits expressed in all leukocytes. LFA-1 is essential ...for neutrophil recruitment to inflamed tissue. Activation of LFA-1 by chemokines allows neutrophils and other leukocytes to undergo arrest, resulting in firm adhesion on endothelia expressing intercellular adhesion molecules (ICAMs). In mice, CXCR2 is the primary chemokine receptor involved in triggering neutrophil arrest, and it does so through "inside-out" activation of LFA-1. CXCR2 signaling induces changes in LFA-1 conformation that are coupled to affinity upregulation of the ligand-binding headpiece (extended with open I domain). Unlike naïve lymphocytes, engagement of P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils stimulates a slow rolling behavior that is mediated by LFA-1 in a distinct activation state (extended with closed I domain). How inside-out signaling cascades regulate the structure and function of LFA-1 is being studied using flow chambers, intravital microscopy, and flow cytometry for ligand and reporter antibody binding. Here, we review how LFA-1 activation is regulated by cellular signaling and ligand binding. Two FERM domain-containing proteins, talin-1 and Kindlin-3, are critical integrin co-activators and have distinct roles in the induction of LFA-1 conformational rearrangements. This review integrates these new results into existing models of LFA-1 activation.
In inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the ...integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III. Both kindlin-3 and talin-1, another FERM domain-containing cytoskeletal protein, are required for integrin activation, but their individual roles in the induction of specific integrin conformers are unclear. Here, we induce differential LFA-1 activation in neutrophils through engagement of the selectin ligand P-selectin glycoprotein ligand-1 or the chemokine receptor CXCR2. We find that talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling, whereas both talin-1 and kindlin-3 are required for induction of the high-affinity conformation of LFA-1 with an open headpiece, which results in neutrophil arrest. In vivo, both slow rolling and arrest are defective in talin-1–deficient neutrophils, whereas only arrest is defective in kindlin-3–deficient neutrophils. We conclude that talin-1 and kindlin-3 serve distinct functions in LFA-1 activation.
Models that postulate the existence of hidden sectors address contemporary questions, such as the source of baryogenesis and the nature of dark matter. Neutron-to-hidden-neutron oscillations are ...among the possible mixing processes and have been tested with ultracold neutron storage and passing-through-wall experiments to set constraints on the oscillation period τ_{nn^{'}}. These searches probe the oscillations as a function of the mass splitting due to the neutron-hidden-neutron energy degeneracy. In this work, we present a new limit derived from neutron disappearance in ultracold neutron beam experiments. The overall limit, given by τ_{nn^{'}}>1 s for |δm|∈2,69 peV(95.45% C.L.), covers the yet unexplored intermediate mass-splitting range and contributes to the ongoing research on hidden sectors.
It has been proposed that there could be a mirror copy of the standard model particles, restoring the parity symmetry in the weak interaction on the global level. Oscillations between a neutral ...standard model particle, such as the neutron, and its mirror counterpart could potentially answer various standing issues in physics today. Astrophysical studies and terrestrial experiments led by ultracold neutron storage measurements have investigated neutron to mirror-neutron oscillations and imposed constraints on the theoretical parameters. Recently, further analysis of these ultracold neutron storage experiments has yielded statistically significant anomalous signals that may be interpreted as neutron to mirror-neutron oscillations, assuming nonzero mirror magnetic fields. The neutron electric dipole moment collaboration performed a dedicated search at the Paul Scherrer Institute and found no evidence of neutron to mirror-neutron oscillations. Thereby, the following new lower limits on the oscillation time were obtained: τnn′>352 s at B′=0 (95% C.L.), τnn′>6s for 0.4μT<B′<25.7μT (95% C.L.), and τnn′/cosβ>9s for 5.0μT<B′<25.4μT (95% C.L.), where β is the fixed angle between the applied magnetic field and the local mirror magnetic field, which is assumed to be bound to the Earth. These new constraints are the best measured so far around B′∼10μT and B′∼20μT.
Techniques for studying the clearance of bacterial infections are critical for advances in understanding disease states, immune cell effector functions, and novel antimicrobial therapeutics. ...Intracellular killing of
by neutrophils can be monitored using a
strain stably expressing GFP, a fluorophore that is quenched when exposed to the reactive oxygen species (ROS) present in the phagolysosome. Here, we expand upon this method by developing a bi-fluorescent
killing assay for use
Conjugating
with a stable secondary fluorescent marker enables the separation of infected cell samples into three populations: cells that have not engaged in phagocytosis, cells that have engulfed and killed
, and cells that have viable internalized
. We identified ATTO647N-NHS Ester as a favorable dye conjugate for generating bi-fluorescent
due to its stability over time and invariant signal within the neutrophil phagolysosome. To resolve the
utility of ATTO647N/GFP bi-fluorescent
, we evaluated neutrophil function in a murine model of chronic granulomatous disease (CGD) known to have impaired clearance of
infection. Analysis of bronchoalveolar lavage (BAL) from animals subjected to pulmonary infection with bi-fluorescent
demonstrated differences in neutrophil antimicrobial function consistent with the established phenotype of CGD.
Abstract Purpose The purpose of the study was threefold: to assess the reliability of shear wave velocities (SWV) measurements in normal skeletal muscles; to evaluate intra- and inter-operator ...reproducibility of measurements for a specific site of the muscle and for the mean value in the whole muscle. Materials and methods Two sets of measurements were performed at three weeks intervals of each other on 16 volunteers by two radiologists on medial gastrocnemius and tibialis anterior muscles. Each muscle was evaluated in 5 different sites, with three measurements for each site in the transverse and longitudinal planes. Reliability of SWV measurements was assessed by means of intraclass correlation coefficient (ICC). Results Reliability of the three independent SWV measurements was excellent, slightly better in the longitudinal plane. Inter/intra-operator reproducibility per site was fair to good in the longitudinal plane and poor to fair in the transverse plane. For global values of the whole muscle, ICC showed good agreement in the longitudinal plane and fair agreement in the transverse plane. Conclusion Quantitative SWV measurements are reliable when performed in rigorous conditions. In conditions that mirror clinical practice, inter/intra-operator reproducibility is moderate, better for longitudinal compared to transverse plane.
Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are ...required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus, neutrophil recruitment needs to be fine-tuned to ensure bacterial killing, while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation, fibrosis, and vascular congestion. Importantly, systemic CLP-induced neutrophil recruitment was attenuated in the lungs, the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils, those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1, ICAM-1, and L-selectin. Interestingly, HS1-deficient neutrophils had similar F-actin content and phagocytic activity, but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection, but insufficient to induce tissue damage.
•HS1 deficiency increases survival during sepsis in mice.•Loss of HS1 decreases neutrophil recruitment.•HS1-deficient neutrophils do not respond to CXCL1 with actin remodeling and cell stiffening.•Lung damage is reduced in septic HS1 deficient mice.•Systemic inflammatory response is reduced without HS1.
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