Biology subjects

Mosher, D. F.

Publications and source records attributed to Mosher, D. F..

5 recordsLinked to original sources

bioRxiv2026

Substance matters: IL5 and IL33 activation of eosinophils on periostin and fibrinogen induce cytoskeletal reorganization and cell death

Human eosinophils activated in suspension with IL5 or IL33 undergo morphological change prior to adhesion. Refractive granules, which contain major basic protein-1 and other toxic proteins, move to one side of the cell, the granulomere, and the two nuclear lobes move to the other. How these features persist when eosinophils become adherent and migrate is not known. We now compare behavior of activated eosinophils on surfaces coated with ITGAM/ITGB2-integrin ligands fibrinogen or periostin using live cell imaging of reporters of tubulin/actin organization and cell viability. We find that unlike eosinophils activated with IL5, IL33-activated eosinophils undergo two stages of activation; a preliminary pear-like activation in which the cell develops polarity, followed by a flattening of the eosinophil into a thin pancake-like morphology with less discrete polarization. IL5-treated eosinophils migrated persistently for more than an hour with nucleopod in the back. In contrast, IL33-treated eosinophils moved more slowly and within 30 min transitioned to a flattened morphology with nuclear lobes in the center and dispersed motile granules. Loss of cell viability after an hour, although variable, in all comparisons was greater among IL33-treated eosinophils on periostin. We sought to understand how cytoskeletal elements may drive these differences in morphology. Cytoskeletal elements had similar responses when activated with IL5/IL33; vimentin collapsed from a web-like network at the periphery of the cell and condensed adjacent to the nucleopod/nuclear interface, f-actin was found in the granulomere as well as the tip of the nucleopod and forward periphery, and microtubules radiated from the microtubule organizing center (MTOC) spanning both the nucleopod and the granulomere. The dynamic formation of microtubules correlated with cellular locomotion, suggesting mesenchymal migration within these cells. These in vitro findings suggest that adhesion plays an important role in determining functional morphology and demonstrates new insights into IL33-activated eosinophils. This work suggests roles for activators and adhesive substrates in regulating the behavior of activated eosinophils in tissues.

cell biology↗

bioRxiv2024

In situ crystalline structure of the Eosinophil major basic protein-1

Eosinophils are white blood cells that participate in innate immune responses and have an essential role in the pathogenesis of inflammatory and neoplastic disorders. Upon activation, eosinophils release cytotoxic proteins such as major basic protein-1 (MBP-1) from cytoplasmic secretory granules (SGr) wherein MBP-1 is stored as nanocrystals. How the MBP-1 nanocrystalline core is formed, stabilized, and subsequently mobilized remains unknown. Here, we report the in-situ structure of crystalline MBP-1 within SGrs of human eosinophils. The structure reveals a mechanism for intragranular crystal packing and stabilization of MBP-1 via a structurally conserved loop region that is associated with calcium-dependent carbohydrate binding in other C-type lectin (CTL) proteins. Single-cell and single-SGr profiling correlating real-space three-dimensional information from cellular montage cryo-electron tomography (cryo-ET) and microcrystal electron diffraction (MicroED) data obtained from non-activated and IL33-activated eosinophils revealed activation-dependent crystal expansion and extrusion of expanded crystals from SGr. These results suggest that MBP-1 crystals play a dynamic role in the release of SGr contents. Collectively, this research demonstrates the importance of in-situ macromolecular structure determination.

biophysics↗

bioRxiv2023

Mutations in Fibronectin Dysregulate Chondrogenesis in Corner Fracture Type Spondylometaphyseal Dysplasia

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER and transitioned into lysosomes. Massive amounts of these vesicles were not cleared from the cytosol. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. FN mutant cells also displayed altered FN splice variants under chondrogenic stimuli. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGF{beta}1) expression, crucial for mesenchymal condensation. Exogenous FN or TGF{beta}1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis. Significance /Highlights* SMDCF-causing mutations in fibronectin impair protein secretion in iPSC-derived mesenchymal stem cells. * Mutant fibronectin and ER protein folding chaperones are directly exported from the rough endoplasmic reticulum into vesicles covered with ribosomes, which transition into lysosomes. * The cells cannot clear the massive accumulation of cytosolic vesicles. * Mutations in fibronectin impair stem cell proliferation, mesenchymal condensation, and the differentiation of MSCs into chondrocytes. * Exogenous addition of purified fibronectin or TGF{beta}-1 improves mesenchymal condensation and chondrogenesis of the FN mutant stem cells.

cell biology↗

bioRxiv2022

Molecular anatomy of eosinophil activation by IL5 and IL33

IL5 and IL33 are major activating cytokines that cause circulating eosinophils to polarize, adhere, and release their granule contents. We correlated microscopic features of purified human blood eosinophils stimulated for 10 min with IL5 or IL33 with phosphoproteomic changes determined by multiplexed isobaric labeling. IL5 caused phosphorylation of sites implicated in JAK/STAT signaling and localization of pYSTAT3 to nuclear speckles whereas IL33 caused phosphorylation of sites implicated in NF{kappa}B signaling and localization of RELA to nuclear speckles. Phosphosites commonly impacted by IL5 and IL33 were involved in networks associated with cytoskeletal organization and eosinophil adhesion and migration. Many differentially regulated phosphosites were in a diverse set of large proteins--RAB44, a "large RAB" associated with crystalloid granules; NHSL2 and VIM that change localization along with the nucleus during polarization; TNFAIP3 vital for control of NF{kappa}B signaling, and SRRM2 and PML that localize, respectively, to nuclear speckles and PML bodies. Gene expression analysis demonstrated differential effects of IL5 and IL33 on IL18, CCL5, CSF1, and TNFSF14. Thus, common effects of IL5 and IL33 on the eosinophil phosphoproteome are important for positioning in tissues, degranulation, and initiation of new protein synthesis whereas specific effects on protein synthesis contribute to phenotypic heterogeneity. KEY POINTSIL33 and IL5 impact common pathways of eosinophil cytoskeletal reorganization, adhesion and migration. Each lobe of the human eosinophil nucleus has a specific anatomy poised for new onset of cytokine-specific transcription and splicing.

cell biology↗

bioRxiv2017

Adaptation Of Staphylococcus saprophyticus At The Host-Pathogen Interface

Human-pathogenic bacteria are found in a variety of niches, including free-living, zoonotic, and microbiome environments. Identifying bacterial adaptions that enable invasive disease is an important means of gaining insight into the molecular basis of pathogenesis and understanding pathogen emergence. Staphylococcus saprophyticus, a leading cause of urinary tract infections, can be found in the environment, food, animals, and the human microbiome. We identified a selective sweep in the gene encoding the Aas adhesin, a key virulence factor that binds host fibronectin. We hypothesize that the mutation under selection (aas_2206A>C) facilitates colonization of the urinary tract, an environment where bacteria are subject to strong shearing forces. The mutation appears to have enabled emergence and expansion of a human pathogenic lineage of S. saprophyticus. These results demonstrate the power of evolutionary genomic approaches in discovering the genetic basis of virulence and emphasize the pleiotropy and adaptability of bacteria occupying diverse niches.\n\nImportanceStaphylococcus saprophyticus is an important cause of urinary tract infections (UTI) in women, which are common, can be severe, and are associated with significant impacts to public health. In addition to being a cause of human UTI, S. saprophyticus can be found in the environment, in food, and associated with animals. After discovering that UTI strains of S. saprophyticus are for the most part closely related to each other, we sought to determine whether these strains are specially adapted to cause disease in humans. We found evidence suggesting that a mutation in the gene aas is advantageous in the context of human infection. We hypothesize that the mutation allows S. saprophyticus to survive better in the human urinary tract. These results show how bacteria found in the environment can evolve to cause disease.

microbiology↗