Electrophysiology of Concatameric Pannexin 1 Channels Reveals the Stoichiometry of C-Terminal Autoinhibition

Codi d'Art Públic: 8008-1 (La República); Reportatge realitzat als dies 4 i 18-7-1990Pericas, Enric (arquitecte); Viaplana, Albert (arquitecte i estructura); Viladomat Massanas, Josep (escultura);Joan Pie (Medalló); Piñón, Helio (Estr

Antineonatal Fc Receptor Antibody Treatment Ameliorates MOG-IgG-Associated Experimental Autoimmune Encephalomyelitis.

BACKGROUND AND OBJECTIVES Myelin oligodendrocyte glycoprotein antibody-associated disorder (MOGAD) is a rare, autoimmune demyelinating CNS disorder, distinct from multiple sclerosis and neuromyelitis optica spectrum disorder. Characterized by pathogenic immunoglobulin G (IgG) antibodies against MOG, a potential treatment strategy for MOGAD is to reduce circulating IgG levels, e.g., by interference with the IgG recycling pathway mediated by the neonatal Fc receptor (FcRn). Although the optic nerve is often detrimentally involved in MOGAD, the effect of FcRn blockade on the visual pathway has not been assessed. Our objective was to investigate effects of a monoclonal anti-FcRn antibody in murine MOG-IgG-associated experimental autoimmune encephalomyelitis (EAE). METHODS We induced active MOG35-55 EAE in C57Bl/6 mice followed by the application of a monoclonal MOG-IgG (8-18C5) 10 days postimmunization (dpi). Animals were treated with either a specific monoclonal antibody against FcRn (α-FcRn, 4470) or an isotype-matched control IgG on 7, 10, and 13 dpi. Neurologic disability was scored daily on a 10-point scale. Visual acuity was assessed by optomotor reflex. Histopathologic hallmarks of disease were assessed in the spinal cord, optic nerve, and retina. Immune cell infiltration was visualized by immunohistochemistry, demyelination by Luxol fast blue staining and complement deposition and number of retinal ganglion cells by immunofluorescence. RESULTS In MOG-IgG-augmented MOG35-55 EAE, anti-FcRn treatment significantly attenuated neurologic disability over the course of disease (mean area under the curve and 95% confidence intervals (CIs): α-FcRn [n = 27], 46.02 [37.89-54.15]; isotype IgG [n = 24], 66.75 [59.54-73.96], 3 independent experiments), correlating with reduced amounts of demyelination and macrophage infiltration into the spinal cord. T- and B-cell infiltration and complement deposition remained unchanged. Compared with isotype, anti-FcRn treatment prevented reduction of visual acuity over the course of disease (median cycles/degree and interquartile range: α-FcRn [n = 16], 0.50 [0.48-0.55] to 0.50 [0.48-0.58]; isotype IgG [n = 17], 0.50 [0.49-0.54] to 0.45 [0.39-0.51]). DISCUSSION We show preserved optomotor response and ameliorated course of disease after anti-FcRn treatment in an experimental model using a monoclonal MOG-IgG to mimic MOGAD. Selectively targeting FcRn might represent a promising therapeutic approach in MOGAD

Synaptotagmin‐7 enhances calcium‐sensing of chromaffin cell granules and slows discharge of granule cargos

Synaptotagmin‐7 (Syt‐7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin‐1 (Syt‐1). Despite a broad appreciation for the importance of Syt‐7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt‐7 (KO cells) and a reconstituted system employing cell‐derived granules expressing either Syt‐7 or Syt‐1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt‐7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt‐7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt‐1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt‐7, granules expressing only Syt‐7 or Syt‐1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt‐7 confers substantially greater calcium sensitivity to granule fusion than Syt‐1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt‐7 plays a central role in regulating secretory output from adrenal chromaffin cells.Syt‐7 is a high‐affinity calcium sensor expressed on chromaffin cell dense core granules. The purpose of this study was to assess the role of Syt‐7 in regulating the secretory response to cholinergic stimulation. Acetylcholine elicits secretion by elevating cytosolic calcium. The calcium sensitivity of exocytosis in cells lacking Syt‐7 is impaired. Cells that lack Syt‐7 also release peptide hormones at faster rates, implicating a role for Syt‐7 in regulating the exocytotic fusion pore. These data demonstrate that Syt‐7 has an important role in triggering exocytosis in cells and is likely to play a role in controlling hormone output, in situ.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/3/jnc14986.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/2/jnc14986-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162737/1/jnc14986_am.pd

SARS-CoV-2 requires acidic pH to infect cells

Publisher Copyright: Copyright © 2022 the Author(s). Published by PNAS.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein–catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.Peer reviewe

Measuring Distances in Supported Bilayers by Fluorescence Interference-Contrast Microscopy: Polymer Supports and SNARE Proteins

Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 ± 1.0 nm. The cleft distance was increased to 3.9 ± 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 ± 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface