Biology subjects

Thomas, M.

Publications and source records attributed to Thomas, M..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Imbalance between IL-36 receptor agonist and antagonist drives neutrophilic inflammation in COPD

Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating novel therapies. Here, we show that COPD patients have increased IL-36{gamma} and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared to control subjects. IL-36{gamma} is derived mainly from small airway epithelial cells (SAEC) and further induced by a viral mimetic, whereas IL-36RA is derived from macrophages. IL-36{gamma} stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36{gamma} thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, that was inhibited by exogenous IL-36RA. The use of a therapeutic antibody that inhibits binding to the IL-36 receptor (IL-36R) attenuated IL-36{gamma} driven inflammation and cellular cross talk. We have demonstrated a novel mechanism for the amplification and propagation of neutrophilic inflammation in COPD and that blocking this cytokine family via a IL-36R neutralising antibody could be a promising new therapeutic strategy in the treatment of COPD.

cell biology↗

bioRxiv2021

Daily ranging and den usage patterns structure fission-fusion dynamics and social associations in spotted hyenas

Environment structure often shapes social interactions. Spatial attractors that draw multiple individuals may play a particularly important role in dispersed groups, where individuals must first encounter one another to interact. We use GPS data recorded simultaneously from five spotted hyenas (Crocuta crocuta) within a single clan to investigate how communal dens and daily ranging patterns shape fission-fusion dynamics (subgroup splits and merges). We introduce a species-general framework for identifying and characterizing dyadic fission-fusion events and describe a taxonomy of ten possible configurations of these events. Applying this framework to the hyena data illuminates the spatiotemporal structure of social interactions within hyenas daily routines. The most common types of fission-fusion events involve close approaches between individuals, do not involve co-travel together, and occur at the communal den. Comparison to permutation-based reference models suggests that den usage structures broad-scale patterns of social encounters, but that other factors influence how those encounters unfold. We discuss the dual role of communal dens in hyenas as physical and social resources, and suggest that dens are an example of a general "social piggybacking" process whereby environmental attractors take on social importance as reliable places to encounter conspecifics, causing social and spatial processes to become fundamentally intertwined.

animal behavior and cognition↗

bioRxiv2021

Educational Attainment Polygenic Scores, Socioeconomic Factors, and Cortical Structure in Children and Adolescents

BackgroundGenome-wide polygenic scores for educational attainment (PGS-EA) and socioeconomic factors, which are correlated with each other, have been consistently associated with academic achievement and general cognitive ability in children and adolescents. Yet, the independent associations of PGS-EA and socioeconomic factors with specific underlying factors at the neural and neurocognitive levels are not well understood. The goal of this study was to examine the unique contributions of PGS-EA and parental education to cortical thickness (CT), cortical surface area (SA), and neurocognitive skills in children and adolescents. MethodsParticipants were typically developing children and adolescents (3-21 years of age; 53% male; N = 391). High-resolution, T1-weighted magnetic resonance imaging data were acquired. PGS-EA were computed based on the most recent genome-wide association study of educational attainment. Sustained attention, inhibitory control, working memory, vocabulary, and episodic memory were measured. ResultsPGS-EA and parental education were independently and significantly associated with SA, vocabulary, and attention outcomes but were not associated with CT. Vertex-wise analyses indicated that higher PGS-EA was significantly associated with greater SA in the left medial orbitofrontal gyrus and inferior frontal gyrus after accounting for parental education. Higher parental education was significantly associated with greater SA in the left parahippocampal gyrus after accounting for PGS-EA. ConclusionsThese findings suggest that education-linked genetics may influence SA, particularly in certain frontal regions, leading to variability in academic achievement. Results suggested genetic confounding in associations between parental education and SA in children and adolescents, with these associations remaining significant after controlling for PGS-EA.

neuroscience↗

bioRxiv2021

Altered interactions between circulating and tissue-resident CD8 T cells with the colonic mucosa define colitis associated with immune checkpoint inhibitors

Therapeutic blockade of co-inhibitory immune receptors PD-1 and CTLA-4 has revolutionized oncology, but treatments are limited by immune-related adverse events (IRAEs). IRAE Colitis (irColitis) is the most common, severe IRAE affecting up to 25% of patients on dual PD-1 and CTLA-4 inhibition. Here, we present a systems biology approach to define the cell populations and transcriptional programs driving irColitis. We collected paired colon mucosal biopsy and blood specimens from 13 patients with irColitis, 8 healthy individuals, and 8 controls on immune checkpoint inhibitors (ICIs), and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis (Luminex). We profiled 299,407 cells from tissue and blood and identified 105 cell subsets that revealed significant tissue remodeling in active disease. Colon mucosal immune populations were dominated by tissue-resident memory (TRM) ITGAE-expressing CD8 T cells representing a phenotypic spectrum defined by gene programs associated with T cell activation, cytotoxicity, cycling, and exhaustion. CD8 TRM and effector CD4 T cells upregulated type 17 immune programs (IL17A, IL26) and Tfh-like programs (CXCL13, PDCD1). We also identified for the first time an increased abundance of two KLRG1 and ITGB2-expressing CD8 T cell populations with circulatory cell markers, including a GZMK TRM-like population and a CX3CR1 population that is predicted to be intravascular. These two populations were more abundant in irColitis patients treated with dual PD-1/CTLA-4 inhibition than those receiving anti-PD-1 monotherapy. They also had significant TCR sharing with PBMCs, suggesting a circulatory origin. In irColitis we observed significant epithelial turnover marked by fewer LGR5-expressing stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs such as the cGAS-STING pathway. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Mesenchymal remodeling was defined by increased endothelial cells, both in irColitis patients and specifically in patients on dual PD-1/CTLA-4 blockade. Cell-cell communication analysis identified putative receptor-ligand pairs that recruit CD8 T cells from blood to inflamed endothelium and positive feedback loops such as the CXCR3 chemokine system that retain cells in tissue. This study highlights the cellular and molecular drivers underlying irColitis and provides new insights into the role of CTLA-4 and PD-1 signaling in maintaining CD8 TRM homeostasis, regulating CD8 T recruitment from blood, and promoting epithelial-immune crosstalk critical to gastrointestinal immune tolerance and intestinal barrier function.

immunology↗

bioRxiv2021

Single cell infection with influenza A virus using drop-based microfluidics

Influenza A virus (IAV) is an RNA virus with high genetic diversity which necessitates the development of new vaccines targeting emerging mutations each year. As IAV exists in genetically heterogeneous populations, current studies focus on understanding population dynamics at the single cell level. These studies include novel methodology that can be used for probing populations at the single cell level, such as single cell sequencing and microfluidics. Here, we introduce a drop-based microfluidics method to study IAV infection at a single cell level by isolating infected host cells in microscale drops. Single human alveolar basal epithelial (A549), Madin-Darby Canine Kidney cells (MDCK) and MDCK + human siat7e gene (Siat7e) cells infected with the pandemic A/California/07/2009 (H1N1) strain were encapsulated within 50 m radii drops and incubated at 37{degrees}C. We demonstrate that drops remain stable over 24 hours, that 75% of cells remain viable, and that IAV virus can propagate within the drops. Drop-based microfluidics therefore enables single cell analysis of viral populations produced from individually infected cells.

microbiology↗

bioRxiv2021

Genome-wide association mapping of ethanol sensitivity in the Diversity Outbred mouse population

BackgroundA strong predictor for the development of alcohol use disorders (AUDs) is altered sensitivity to the intoxicating effects of alcohol. Individual differences in the initial sensitivity to alcohol are controlled in part by genetic factors. Mice offer a powerful tool for elucidating the genetic basis of behavioral and physiological traits relevant to AUDs; but conventional experimental crosses have only been able to identify large chromosomal regions rather than specific genes. Genetically diverse, highly recombinant mouse populations allow for the opportunity to observe a wider range of phenotypic variation, offer greater mapping precision, and thus increase the potential for efficient gene identification. MethodsWe have taken advantage of the Diversity Outbred (DO) mouse population to identify and precisely map quantitative trait loci (QTL) associated with ethanol sensitivity. We phenotyped 798 male J:DO mice for three measures of ethanol sensitivity: ataxia, hypothermia, and loss of the righting response. We used high density MEGAMuga and GIGAMuga arrays to obtain genotypes ranging from 77,808 - 143,259 SNPs. In addition, we performed RNA sequencing in striatum to map expression QTLs and to identify gene expression-trait correlations. ResultsWe then applied a systems genetic strategy to identify narrow QTLs and construct the network of correlations that exist between DNA sequence, gene expression values and ethanol-related phenotypes to prioritize our list of positional candidate genes. ConclusionsOur results can be used to identify alleles that contribute to AUDs in humans, elucidate causative biological mechanisms, or assist in the development of novel therapeutic interventions.

genetics↗

bioRxiv2021

A non-functional copy of the salmonid sex determining gene (sdY) is responsible for the "apparent" XY females in Chinook salmon, Oncorhynchus tshawytscha.

Many salmonids have a male heterogametic (XX/XY) sex determination system, and they are supposed to have a conserved master sex determining gene (sdY), that interacts at the protein level with Foxl2 leading to the blockage of the synergistic induction of Foxl2 and Nr5a1 of the cyp19a1a promoter. However, this hypothesis of a conserved master sex determining role of sdY in salmonids is still challenged by a few exceptions, one of them being the presence of some naturally occurring "apparent" XY Chinook salmon females. Here we show that XY Chinook salmon females have a sdY gene (sdY-N183), which has one missense mutation leading to a substitution of a conserved isoleucine to an asparagine (SdY I183N). In contrast, Chinook salmon males have both a non-mutated sdY-I183 gene and the missense mutation sdY-N183 gene. The 3D model of SdY-N183 predicts that the I183N hydrophobic to hydrophilic amino acid change leads to a local modification of the {beta}-sandwich structure of SdY. Using in vitro cell transfection assays we found that SdY-N183, like SdY-I183, is preferentially localized in the cytoplasm. However, compared to SdY-I183, SdY-N183 is more prone to degradation, its nuclear translocation by Foxl2 is reduced and SdY-N183 is unable to significantly repress the synergistic Foxl2/Nr5a1 induction of the cyp19a1a promoter. Altogether our results suggest that the sdY-N183 gene of XY Chinook females is a non-functional gene and that SdY-N183 is no longer able to promote testicular differentiation by impairing the synthesis of estrogens in the early differentiating gonads of wild Chinook salmon XY females.

genetics↗

bioRxiv2021

Exosomes from adipose-derived stem cells alleviate myocardial infarction via microRNA-31/FIH1/HIF-1α pathway

Our previous study has revealed that exosomes from adipose-derived stem cells (ASCs) promote angiogenesis in subcutaneously transplanted gels by delivery of microRNA-31 (miR-31) which targets factor inhibiting hypoxia-inducible factor-1 (FIH1) in recipient cells. Here we hypothesized that ASC exosomes alleviate ischemic diseases through miR-31/FIH1/hypoxia-inducible factor-1 (HIF-1) signaling pathway. Exosomes from ASCs were isolated by sequential centrifugations, and were characterized with nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting analysis for exosomal markers. Results from laser imaging of ischemic mouse hindlimb revealed that exosomes enhanced the blood perfusion, and this enhancement was impaired when using miR-31-depleted exosomes. Immunohistochemistry analysis showed that administration of exosomes resulted in a higher arteriole density and larger CD31+ area in ischemic hindlimb than miR-31-delpleted exosomes. Similarly, knockdown of miR-31 in exosomes reduced the effects of the exosomes on increasing ventricular fraction shortening and CD31+ area, and on decreasing infarct size. Exosomes promoted endothelial cell migration and tube formation. These changes were attenuated when miR-31 was depleted in the exosomes or when FIH1 was overexpressed in the endothelial cells. Furthermore, the results from co-immunoprecipitation and luciferase reporter assay demonstrated that the effects of exosomes on elevating the binding of HIF-1 with co-activator p300 and enhancing HIF-1 activity were decreased when miR-31 was depleted in the exosomes or FIH1 was overexpressed. Our findings provide evidence that exosomes from ASCs promote angiogenesis in both mouse ischemic hindlimb and heart through transport of miR-31 which targets FIH1 and therefore triggers HIF-1 transcriptional activation.

molecular biology↗

bioRxiv2021

A toxic friend: Genotoxic and mutagenic activity of the probiotic strain Escherichia coli Nissle 1917

The probiotic Escherichia coli strain Nissle 1917 (DSM 6601, Mutaflor), generally considered as beneficial and safe, has been used for a century to treat various intestinal diseases. However, Nissle 1917 hosts in its genome the pks pathogenicity island that codes for the biosynthesis of the genotoxin colibactin. Colibactin is a potent DNA alkylator, suspected to play a role in colorectal cancer development. We show in this study that Nissle 1917 is functionally capable of producing colibactin and inducing interstrand crosslinks in the genomic DNA of epithelial cells exposed to the probiotic. This toxicity was even exacerbated with lower doses of the probiotic, when the exposed cells started to divide again but exhibited aberrant anaphases and increased gene mutation frequency. DNA damage was confirmed in vivo in mouse models of intestinal colonization, demonstrating that Nissle 1917 produces the genotoxin in the gut lumen. Although it is possible that daily treatment of adult humans with their microbiota does not produce the same effects, administration of Nissle 1917 as a probiotic or as a chassis to deliver therapeutics might exert long term adverse effects and thus should be considered in a risk versus benefit evaluation. ImportanceNissle 1917 is sold as a probiotic and considered safe even though it is known since 2006 that it encodes the genes for colibactin synthesis. Colibactin is a potent genotoxin that is now linked to causative mutations found in human colorectal cancer. Many papers concerning the use of this strain in clinical applications ignore or elude this fact, or misleadingly suggest that Nissle 1917 does not induce DNA damage. Here, we demonstrate that Nissle 1917 produces colibactin in vitro and in vivo and induces mutagenic DNA damage. This is a serious safety concern that must not be ignored, for the interests of patients, the general public, health care professionals and ethical probiotic manufacturers.

microbiology↗

bioRxiv2021

Sex-specific interactions between procedural and deliberative decision-making systems in a mouse model of Alzheimer's disease

A central question in aging and Alzheimers disease (AD) is when and how neural substrates underlying decision-making are altered. Here we show that while APP mice, a commonly used mouse model of AD, were able to learn Restaurant Row, a complex neuroeconomic decision-making task, they were significantly impaired in procedural, habit-forming, aspects of cognition and relied heavily on deliberation when making decisions. Surprisingly, these behavioral changes are associated with amyloid-beta (A{beta}) pathology and network remodeling in the striatum, a key brain region involved in procedural cognition. Furthermore, APP mice and control mice relied on distinct sex-specific strategies in this neuroeconomic task. These findings provide foundational pillars to examine how aging and age-related neurodegenerative diseases impact decision-making across sexes. They also highlight the need for complex behavioral tasks that allow for the dissociation of competing neurally-distinct decision-making circuits to get an accurate picture of changes in neurodegenerative models of human disease.

neuroscience↗

bioRxiv2021

CropMetaPop: a population genetics program to simulate the evolution of crop metapopulations

Stochastic simulation programs are useful to study the evolution of crop populations in changing environments. These programs require to take into account some specific features characterizing farmers practices such as seed circulation or seed mixing. However such human-mediated evolutionary processes are different from the classical colonization or migration models used in most existing stochastic simulation programs for quantitative population genetics. To fill this gap, CropMetaPop is an individual based forward-time simulation program for quantitative population genetics developed to represent a diversity of socially established rules underlying seed circulation between farmers. For instance, one farmer can give seeds to neighbours according to his stock and receive from them according to his needs. Crop-Metapop also takes into account other important evolutionary forces encountered in crop diversity management systems, such as different population sizes and selection modalities for different populations. CropMetaPop makes it possible to study the impacts of different crop biodiversity management strategies. For this purpose, it simulates the genetic evolution over time of several crop populations submitted to genetic drift and selection and linked by explicitly modeled seed circulation and mixtures.

evolutionary biology↗

bioRxiv2020

Nature of active forces in tissues: how contractile cells can form extensile monolayers

Actomyosin machinery endows cells with contractility at a single cell level. However, at a tissue scale, cells can show either contractile or extensile behaviour based on the direction of pushing or pulling forces due to neighbour interactions or substrate interactions. Previous studies have shown that a monolayer of fibroblasts behaves as a contractile system1 while a monolayer of epithelial cells2,3 or neural crest cells behaves as an extensile system.4 How these two contradictory sources of force generation can coexist has remained unexplained. Through a combination of experiments using MDCK (Madin Darby Canine Kidney) cells, and in-silico modeling, we uncover the mechanism behind this switch in behaviour of epithelial cell monolayers from extensile to contractile as the weakening of intercellular contacts. We find that this switch in active behaviour also promotes the buildup of tension at the cell-substrate interface through an increase in actin stress fibers and higher traction forces. This in turn triggers a mechanotransductive response in vinculin translocation to focal adhesion sites and YAP (Yes-associated protein) transcription factor activation. Our studies also show that differences in extensility and contractility act to sort cells, thus determining a general mechanism for mechanobiological pattern formation during cell competition, morphogenesis and cancer progression.

biophysics↗

bioRxiv2020

The microbiota plays a critical role in the reactivity of lung immune components to innate ligands

The microbiota contributes to shaping efficient and safe immune defenses in the gut. However, little is known about the role of the microbiota in the education of pulmonary innate immune responses. Here, we tested whether the endogenous microbiota can modulate reactivity of pulmonary tissue to pathogen stimuli by comparing the response of specific pathogen-free (SPF) and germ-free (GF) mice. Using SPF and GF mice intranasally exposed to lipopolysaccharide (LPS), a component of Gram-negative bacteria, we observed earlier and greater inflammation in the pulmonary compartment of GF mice than that of SPF mice. Toll-like receptor 4 (TLR4) was more abundantly expressed in the lungs of GF mice than those of SPF mice at steady state, which could predispose the innate immunity of GF mice to strongly react to environmental stimuli. Lung explants were stimulated with different TLR agonists or infected with the human airways pathogen, respiratory syncytial virus (RSV), resulting in greater inflammation under almost all conditions for the GF explants. Finally, alveolar macrophages (AM) from GF mice presented a higher innate immune response upon RSV infection than those of SPF mice. Overall, these data suggest that the presence of microbiota in SPF mice induced a process of innate immune tolerance in the lungs by a mechanism which remains to be elucidated. Our study represents a step forward to establishing the link between the microbiota and the immune reactivity of the lungs. Plain Language summaryMicrobiota represents an important partner of immunologic system at the interface between immune cells and epithelium. It is well known, notably in the gut, that the microbiota contributes in shaping efficient and safe defenses. However, little is known about the role of the microbiota in the education of pulmonary innate immune responses. In this study, we postulate that endogenous microbiota could dampen an excessive reactivity of pulmonary tissue to external stimuli. Thus, we sought to study the innate immune reaction switched on by viral or bacterial ligands in respiratory tract cells coming from mice with or without microbiota (germ-free condition, GF). Altogether, our results show a higher inflammatory reaction in GF condition. This study represents a step forward to better establish the link between the microbiota and the reactivity of the lung tissue. Not only these data demonstrate that the microbiota educates the pulmonary innate immune system, but also contributes the emerging concept of using respiratory commensal bacteria as potential next-generation probiotics to prevent susceptibility to respiratory diseases.

immunology↗

bioRxiv2020

Non-thalamic origin of zebrafish sensory relay nucleus: convergent evolution of visual pathways in amniotes and teleosts

Ascending visual projections similar to the mammalian thalamocortical pathway are found in a wide range of vertebrate species, but their homologous relationship is debated. To get better insights into their evolutionary origin, we examined the developmental origin of a visual relay nucleus in zebrafish (a teleost fish). Similarly to the tectofugal visual thalamic nuclei in amniotes, the lateral part of the preglomerular complex (PG) in teleosts receives tectal information and projects to the pallium. However, our cell lineage study reveals that the majority of PG cells are derived from the midbrain, not from the forebrain. We also demonstrate that the PG projection neurons develop gradually until juvenile stage, unlike the thalamic projection neurons. Our data suggest that teleost PG is not homologous to the amniote thalamus and that thalamocortical-like projections can evolve from a non-forebrain cell population. Thus, sensory pathways in vertebrate brains exhibit a surprising degree of variation.

neuroscience↗

bioRxiv2020

C-Eci: A Cubic-Eci Combined Clearing Method For 3D Follicular Content Analysis In The Fish Ovary

Deciphering mechanisms of oocyte development in female fishes still remains challenging and a comprehensive overview of this process at the level of the organ is still needed. The recent development optical tissue clearing methods have tremendously boosted the 3D imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the non-toxic solvent-based Eci method for efficient clearing and the CUBIC method to enhance lipid removal and reduce non-specific staining. The methyl green fluorescent dye was used to stain nuclei and delineate follicles. Using this procedure (named C-Eci), ovaries of both medaka and trout could be imaged in 3D and all follicles analyzed. To our knowledge this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-Eci methods thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further morphological studies. SummaryA modified ethyl-cinnamate-based clearing method allows solving fish ovary-specific challenges for 3D imaging, including high lipid-contents, and analyzing the ovarian follicular content in medaka and trout.

developmental biology↗

bioRxiv2019

Supporting Computational Apprenticeship through educational and software infrastructure. A case study in a mathematical oncology research lab

There is growing awareness of the need for mathematics and computing to quantitatively understand the complex dynamics and feedbacks in the life sciences. Although several institutions and research groups are conducting pioneering multidisciplinary research, communication and education across fields remains a bottleneck. The opportunity is ripe for using education research-supported mechanisms of cross-disciplinary training at the intersection of mathematics, computation and biology. This case study uses the computational apprenticeship theoretical framework to describe the efforts of a computational biology lab to rapidly prototype, test, and refine a mentorship infrastructure for undergraduate research experiences. We describe the challenges, benefits, and lessons learned, as well as the utility of the computational apprenticeship framework in supporting computational/math students learning and contributing to biology, and biologists in learning computational methods. We also explore implications for undergraduate classroom instruction, and cross-disciplinary scientific communication.

scientific communication and education↗

bioRxiv2019

Minimal genome-wide human CRISPR-Cas9 library

CRISPR guide-RNA libraries have been iteratively optimised to provide increasingly efficient reagents, although their large size is a barrier for many applications. We designed an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9), by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other libraries while preserving assay sensitivity and specificity. MinLibCas9 increases the dynamic range of CRISPR-Cas9 loss-of-function screens and extends their application to complex models and assays.

genomics↗

bioRxiv2019

Desmosomal coupling in apoptotic cell extrusion

The mechanical coupling of epithelia enables coordination of tissue functions and collective tissue movements during different developmental and physiological processes. This coupling is ensured by cell-cell junctions, including adherens junctions (AJs) and desmosomal junctions (DJs) [1, 2]. During apoptosis, or programmed cell death, a dead cell is expelled from the tissue by coordinated processes between the dying cell and its neighbors. Apoptotic cell extrusion is driven by actomyosin cable formation and its contraction, and lamellipodial crawling of the neighboring cells (Fig. S1A-A, Movie S1) [3-6]. Throughout cell extrusion, the mechanical coupling of epithelia needs to be maintained in order to preserve tissue homeostasis [3]. Although much is known about the regulation of AJs in apoptotic cell extrusion [6-9], the role and dynamics of DJs during this process remains poorly understood. Here, we show that DJs stay intact throughout and are crucial for apoptotic cell extrusion. Pre-existing DJs between the apoptotic cell and neighboring non-dying cells remain intact even during the formation of de novo DJs between non-dying cells, suggesting that the neighboring cells possess two DJs in the middle of apoptotic cell extrusion. We further found that an actomyosin cable formed in the vicinity of DJs upon apoptosis, and subsequently deviated from DJs during its constriction. Interestingly, the departure of the actomyosin cable from DJs coincided with the timing when DJs lost their straightness, suggesting a release of junctional tension at DJs, and a mechanical coupling between DJs and actomyosin contractility. The depletion of desmoplakin, which links desmosomes and intermediate filaments, resulted in defective apical contraction and an inability to form de novo DJs, leading to a failure of apoptotic cell extrusion. Our study provides a framework to explain how desmosomes play pivotal roles in maintaining epithelial sheet integrity during apoptotic cell extrusion.\n\nO_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC=\"FIGDIR/small/816280v1_figs1.gif\" ALT=\"Figure 1\">\nView larger version (49K):\norg.highwire.dtl.DTLVardef@454179org.highwire.dtl.DTLVardef@cd7e75org.highwire.dtl.DTLVardef@1295ed1org.highwire.dtl.DTLVardef@1102c8f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure S1:C_FLOATNO Desmosomal junctions at the interface during apoptotic extrusion. A) Confocal images showing actin (Lifeact-Ruby) dynamics during apoptotic cell extrusion in both apical (top panel) and basal (bottom panel) sections of the neighboring cell. Blue-filled and open arrows show the actomyosin cable (top panel) and the lamellipodia (bottom panel) respectively. A) Illustration showing the definition of interface ROI during extrusion. A) Graphs plotting normalized apoptotic cell area (black) and actin (blue) intensity at the dying cell interface during extrusion (n=5 independent experiments). B) Confocal images showing the localization of overexpressed Dsg-FLAG-EGFP (green) and endogenous desmoplakin (red) in MDCK monolayers (n=3 independent staining experiments). B) Magnified images of the white-filled and dotted boxes from B showing co-existence of dotted and continuous junctional clusters in the cell. C) Confocal images showing mCherry-E-cadherin dynamics at the interface during extrusion. C) Graphs plotting normalized apoptotic cell area (black) and E-Cadherin (blue) intensity during constriction (n=5 independent experiments). D) Confocal images showing desmoglein 2 and membrane (mCherry-delta-PH) dynamics in both apical (top panel) and basal (bottom panel) sections of the cell. White arrows (bottom panel) highlights formation of de novo junctions (n=8 independent experiments). E) Confocal images of endogenous desmosomes in the apical (top panel) and basal (bottom panel) sections during different phases of cell extrusion. Yellow and white arrows indicate pre-existing and de novo junctions, respectively (n=182 extrusion events from 7 independent experiments). F) Confocal images showing desmosome (green), killer red (mem-Killer red, cyan) and caspase 3 (magenta) before and after photo-irradiation. White arrow denotes extent of the dying cell. F) Confocal images showing desmosome dynamics (black) in the apical (top panel) and basal (bottom panel) sections of the neighboring cell. Red and blue arrows highlight pre-existing and de novo junctions respectively. F) Transverse view of a neighboring cell showing DJ dynamics after apoptosis induction through Killer Red. Red and blue arrows indicate pre-existing and de novo junctions respectively (n=4 independent experiments). The data from A and C represent mean {+/-}SEM. Scale bars, 10 m except C (5 m).\n\nC_FIG

cell biology↗