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Energy deficiency selects crowded live epithelial cells for extrusion.
No full textEpithelial cells work collectively to provide a protective barrier, yet they turn over rapidly through cell division and death. If the numbers of dividing and dying cells do not match, the barrier can vanish, or tumours can form. Mechanical forces through the stretch-activated ion channel Piezo1 link both of the processes; stretch promotes cell division, whereas crowding triggers live cells to extrude and then die1,2. However, it was not clear what selects a given crowded cell for extrusion. Here we show that the crowded cells with the least energy and membrane potential are selected for extrusion. Crowding triggers sodium (Na+) entry through the epithelial Na+ channel (ENaC), which depolarizes cells. While those with sufficient energy repolarize, those with limited ATP remain depolarized, which, in turn, triggers water egress through the voltage-gated potassium (K+) channels Kv1.1 and Kv1.2 and the chloride (Cl-) channel SWELL1. Transient water loss causes cell shrinkage, amplifying crowding to activate crowding-induced live cell extrusion. Thus, our findings suggest that ENaC acts as a tension sensor that probes for cells with the least energy to extrude and die, possibly damping inadvertent crowding activation of Piezo1 in background cells. We reveal crowding-sensing mechanisms upstream of Piezo1 that highlight water regulation and ion channels as key regulators of epithelial cell turnover
Hybrid dark-field and attenuation contrast retrieval for laboratory-based X-ray tomography.
No full textX-ray dark-field imaging highlights sample structures through contrast generated by sub-resolution features within the inspected volume. Quantifying dark-field signals generally involves multiple exposures for phase retrieval, separating contributions from scattering, refraction, and attenuation. Here, we introduce an approach for non-interferometric X-ray dark-field imaging that presents a single-parameter representation of the sample. This fuses attenuation and dark-field signals, enabling the reconstruction of a unified three-dimensional volume. Notably, our method can obtain dark-field contrast from a single exposure and employs conventional back projection algorithms for reconstruction. Our approach is based on the assumption of a macroscopically homogeneous material, which we validate through experiments on phantoms and on biological tissue samples. The methodology is implemented on a laboratory-based, rotating anode X-ray tube system without the need for coherent radiation or a high-resolution detector. Utilizing this system with streamlined data acquisition enables expedited scanning while maximizing dose efficiency. These attributes are crucial in time- and dose-sensitive medical imaging applications and unlock the ability of dark-field contrast with high-throughput lab-based tomography. We believe that the proposed approach can be extended across X-ray dark-field imaging implementations beyond tomography, spanning fast radiography, directional dark-field imaging, and compatibility with pulsed X-ray sources
Precision probing of O-GalNAc glycosylation using bump-and-hole engineering.
No full textGlycosylation is a profound influencer of glycoprotein function. Glycans have a critical impact on health and disease, yet the tools to study them have trailed behind proteins and nucleic acids. O-GalNAc glycosylation involves the addition of N-acetylgalactosamine (GalNAc) to protein substrates. Dysregulation of O-GalNAc glycosylation is implicated in many pathologies such as cancer. Studying O-GalNAc glycosylation is complicated by the lack of a consensus sequence for initiation and the complex interdependence between a large family of 20 GalNAc transferases (GalNAc-Ts) in human cells. These issues necessitate precise methods of interrogating enzyme function. Herein, we discuss our own advances into the generation of precision tools to study O-GalNAc glycosylation and other glycosylation types. We discuss the use of bump-and-hole engineering to illuminate the roles of individual GalNAc-Ts. Engineering biosynthetic pathways enables cell line-specific uptake of chemical, editable sugars in co-culture settings. We provide an insight into the state-of-the-art in this field
Mycobacterium tuberculosis phagosome Ca2+ leakage triggers multimembrane ATG8/LC3 lipidation to restrict damage in human macrophages.
No full textThe role of canonical autophagy in controlling Mycobacterium tuberculosis (Mtb), referred to as xenophagy, is understood to involve targeting Mtb to autophagosomes, which subsequently fuse with lysosomes for degradation. Here, we found that Ca2+ leakage after Mtb phagosome damage in human macrophages is the signal that triggers autophagy-related protein 8/microtubule-associated proteins 1A/1B light chain 3 (ATG8/LC3) lipidation. Unexpectedly, ATG8/LC3 lipidation did not target Mtb to lysosomes, excluding the canonical xenophagy. Upon Mtb phagosome damage, the Ca2+ leakage-dependent ATG8/LC3 lipidation occurred on multiple membranes instead of single or double membranes excluding the noncanonical autophagy pathways. Mechanistically, Ca2+ leakage from the phagosome triggered the recruitment of the V-ATPase-ATG16L1 complex independently of FIP200, ATG13, and proton gradient disruption. Furthermore, the Ca2+ leakage-dependent ATG8/LC3 lipidation limited Mtb phagosome damage and restricted Mtb replication. Together, we uncovered Ca2+ leakage as the key signal that triggers ATG8/LC3 lipidation on multiple membranes to mitigate Mtb phagosome damage
From cytokines to tuberculosis and back: My journey to understanding the immune response to infection.
No full textI felt honored by the invitation to write this autobiography, although it was an arduous task to describe my journey through science: first bacterial adhesion, then cytokine function, and then immune responses in tuberculosis. Since only seven women had been authors of autobiographies for the Annual Review of Immunology, I felt I couldn't refuse to contribute to Volume 43 of the journal. Moreover, this was a good occasion to record my appreciation to all the lab members and collaborators for their contributions over the last 40 years, to remember the exciting times, and to reflect on the obstacles we faced. I often reflect on this line that is commonly attributed to Winston Churchill: Success is not final; failure is not fatal: It is the courage to continue that counts. What kept me going was a burning desire to know how things work and find enjoyment in the discovery. This passion to understand immune responses to infection remains with me to this day. I thank all those I have interacted with for the support and friendship they provided
A Drosophila holidic diet optimized for growth and development.
No full textDiets composed of chemically pure components (holidic diets) are useful for determining the metabolic roles of individual nutrients. For the model organism Drosophila melanogaster, existing holidic diets are unable to support the rapid growth characteristic of the larval stage. Here, we use a nutrient co-optimization strategy across more than 50 diet variants to design a holidic diet for fast development (HolFast), a holidic medium tailored for fast larval growth and development. We identify dietary amino acid ratios optimal for developmental speed but show that they compromise survival unless vitamins and sterols are co-optimized. Rapid development on HolFast is not improved by adding fatty acids, but it is dependent upon their de novo synthesis in the fat body via fatty acid synthase (FASN). HolFast outperforms other holidic diets, supporting rates of growth and development close to those of yeast-based diets and, under germ-free conditions, identical. HolFast has wide applications in nutritional and metabolic studies of Drosophila development
Expedited SARS‐CoV‐2 main protease inhibitor discovery through modular ‘direct‐to‐biology’ screening
No full textReactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low‐throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for ‘direct‐to‐biology’ (D2B) screening of cysteine‐targeting chloroacetamide RFs, wherein synthesis is performed in 384‐well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS‐CoV‐2 main protease (MPro), an established drug target for the treatment of COVID‐19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a ‘switch’ to a reversible inhibitor series. This example of ligand discovery for MPro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non‐covalent ligands
The glia-immune network: Astrocytes and oligodendrocytes as microglial co-ordinators in health and disease.
No full textIt has long been established that microglia are integral to the CNS immune system. Their surveying and adaptive nature is key in brain development and maintaining homeostasis as well as in the manifestation and progression of neuropathology. However with advancing technology it is becoming increasingly recognised that they do not serve this role in isolation. Previously most work has focused on microglia-derived signalling, with less attention on the sensing and signalling capacity of macroglia (astrocytes, oligodendrocytes). Recent developments in single-cell transcriptomics have allowed extensive analysis of cell profiles in health and disease; these studies have drawn attention to the capacity of macroglia to also engage in immune signalling pathways. This is particularly relevant in neuropathologies, including in Alzheimer's disease (AD), where specific disease-associated profiles of glia (DAGs) have been established. These changes are predominantly related to immune pathways, which were long considered limited to immune cells, including cytokine and chemokine production, antigen presentation and phagocytosis. There is an increasing body of evidence that glia should be considered as active components of the CNS immune system forming a glia-specific immune-like network, whereby macroglia, acting as sensors of the CNS microenvironment, function within this network to co-ordinate diverse CNS effect(s)/function(s). To gain an in-depth understanding of AD pathology, the intimate molecular dialogue of glia needs to be elucidated. This review aims to examine the evidence for macroglia-derived immune signalling and its relevance in health and disease
PEPSeek-mediated identification of novel epitopes from viral and bacterial pathogens and the impact on host cell immunopeptidomes.
No full textHere, we develop PEPSeek, a web-server based software to allow higher performance in the identification of pathogen-derived epitope candidates detected via mass spectrometry in MHC class I immunopeptidomes. We apply it to human and mouse cell lines infected with either SARS-CoV-2, Listeria monocytogenes or Chlamydia trachomatis, thereby identifying a large number of novel antigens and epitopes that we prove to be recognized by CD8+ T cells. In infected cells, we identified antigenic peptide features that suggested how processing and presentation of pathogenic antigens differ between pathogens. The quantitative tools of PEPSeek also helped to define how C. trachomatis infection cycle could impact on the antigenic landscape of the host human cell system, likely reflecting metabolic changes occurred in the infected cells.</p
WNK1-dependent water influx is required for CD4+ T cell activation and T cell-dependent antibody responses.
No full textSignaling from the T cell antigen receptor (TCR) on CD4+ T cells plays a critical role in adaptive immune responses by inducing T cell activation, proliferation, and differentiation. Here we demonstrate that WNK1, a kinase implicated in osmoregulation in the kidney, is required in T cells to support T-dependent antibody responses. We show that the canonical WNK1-OXSR1-STK39 kinase signaling pathway is required for TCR signaling in CD4+ T cells, their subsequent entry into the cell cycle, and suppression of the ATR-mediated G2/M cell cycle checkpoint. We show that the WNK1 pathway regulates ion influx leading to water influx, potentially through AQP3, and that water influx is required for TCR-induced signaling and cell cycle entry. Thus, TCR signaling via WNK1, OXSR1, STK39 and AQP3 leads to water entry that is essential for CD4+ T cell proliferation and hence T cell-dependent antibody responses