Biology subjects

Webb, S.

Publications and source records attributed to Webb, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Beta-hairpin Mechanism of Autoinhibition and Activation in the Kinesin-2 Family

Members of the kinesin-2 family coordinate with other motors to power diverse physiological processes, but the structural mechanisms regulating kinesin-2 activity have been unknown. Distinctively, kinesin-2s canonically function as heterotrimers of two different motor subunits (e.g. Kif3A and Kif3B) and Kap3, but the role of heterotrimerization has yet to fully emerge. Here, we combine structural, cell biological, and single-molecule approaches to dissect kinesin-2 regulation as a heterodimer, heterotrimer, and quaternary complex with a cargo adaptor (APC). We identify a conserved motif in the tail of kinesin-2s (the beta-hairpin motif) that controls kinesin-2 motility by sequestering the motor domains away from their microtubule track, using striking molecular mimicry. Our data reveal how Kap3 binds via a multipartite interface with Kif3A and Kif3B, and - rather than activating motility directly - provides a platform on which cargo adaptors can engage and occlude the beta-hairpin motif. Together, these data articulate a structural framework for kinesin-2 activation, coordination with dynein, and adaptation for different biological functions.

cell biology↗

bioRxiv2024

Eye-movement artifact correction in infant EEG

BackgroundIndependent Component Analysis (ICA) is a well-established approach to clean EEG and remove the impact of signals of non-neural origin, such as those from muscular activity and eye movements. However, evidence suggests that ICA removes artifacts less effectively in infants than in adults. This study systematically compares ICA and Artifact Blocking (AB), an alternative approach proposed to improve eye-movement artifact correction in infant EEG. MethodsWe analyzed EEG collected from 50 infants between 6 and 18 months of age as part of the International Infant EEG Data Integration Platform (EEG-IP), a longitudinal multi-study dataset. EEG was recorded while infants sat on their caregivers laps and watched videos. We used ICA and AB to correct for eye-movement artifacts in the EEG and calculated the proportion of effectively corrected segments, signal-to-noise ratio (SNR), power-spectral density (PSD), and multiscale entropy (MSE) in manually selected EEG segments with and without eye-movement artifacts. ResultsOn the one hand, the proportion of effectively corrected segments indicated that ICA corrected eye-movement artifacts (sensitivity) better than AB. SNR and PSD indicated that both AB and ICA correct eye-movement artifacts with equal sensitivity. MSE gave mixed results. On the other hand, AB caused less distortion to the clean segments (specificity) for SNR, PSD, and MSE. ConclusionsOur results suggest that ICA is more sensitive (i.e., it better removes artifacts) but less specific (it distorts clean signals) than AB for correcting eye-movement artifacts in infant EEG.

neuroscience↗

bioRxiv2023

Single cell transcriptomics reveals chondrocyte differentiation dynamics in vivo and in vitro

The consistent production of in vitro chondrocytes that faithfully recapitulate in vivo development would be of great benefit for musculoskeletal disease modelling and regenerative medicine. Current efforts are often limited by off-target differentiation, resulting in a heterogeneous product. Furthermore, the lack of comparison to human embryonic tissue, precludes detailed evaluation of in vitro cells. Here, we perform single-cell RNA sequencing of embryonic long bones dissected from first trimester hind limbs from a range of gestational ages. We combine this with publicly available data to form a detailed atlas of endochondral ossification, which we then use to evaluate a series of published in vitro chondrogenesis protocols, finding substantial variability in cell states produced by each. We apply single-nuclear RNA sequencing to one protocol to enable direct comparison between in vitro and in vivo, and perform trajectory alignment between the two to reveal differentiation dynamics at the single-cell level, shedding new light on off-target differentiation in vitro. Using this information, we inhibit the activity of FOXO1, a transcription factor predicted to be active in embryonic bone development and in chondrogenic cells in vitro, and increase chondrocyte transcripts in vitro. This study therefore presents a new framework for evaluating tissue engineering protocols, using single-cell data from human development to drive improvement and bring the prospect of true engineered cartilage closer to reality.

bioengineering↗

bioRxiv2023

Effects of TP63 Mutations on Keratinocyte Adhesion and Migration

The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC.

cell biology↗

bioRxiv2023

Automatic cell type harmonization and integration across Human Cell Atlas datasets

Harmonizing cell types across the single-cell community and assembling them into a common framework is central to building a standardized Human Cell Atlas. Here we present CellHint, a predictive clustering tree-based tool to resolve cell type differences in annotation resolution and technical biases across datasets. CellHint accurately quantifies cell-cell transcriptomic similarities and places cell types into a relationship graph that hierarchically defines shared and unique cell subtypes. Application to multiple immune datasets recapitulates expert-curated annotations. CellHint also reveals underexplored relationships between healthy and diseased lung cell states in eight diseases. Furthermore, we present a workflow for fast cross-dataset integration guided by harmonized cell types and cell hierarchy, which uncovers underappreciated cell types in adult human hippocampus. Finally, we apply CellHint to 12 tissues from 38 datasets, providing a deeply curated cross-tissue database with [~]3.7 million cells and various machine learning models for automatic cell annotation across human tissues.

bioinformatics↗

bioRxiv2023

Histone divergence in Trypanosoma brucei results in unique alterations to nucleosome structure

Eukaryotes have a multitude of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically and structurally characterised nucleosome core particles (NCPs) from the kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened overall DNA binding. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.

biochemistry↗

bioRxiv2022

Multi-organ functions of yolk sac during human early development

The yolk sac (YS) represents an evolutionarily-conserved extraembryonic structure that ensures timely delivery of nutritional support and oxygen to the developing embryo. However, the YS remains ill-defined in humans. We therefore assemble a complete single cell 3D map of human YS from 3-8 post conception weeks by integrating multiomic protein and gene expression data. We reveal the YS as a site of primitive and definitive haematopoiesis including a YS-specific accelerated route to macrophage production, a source of nutritional/metabolic support and a regulator of oxygen-carrying capacity. We reconstruct the emergence of primitive haematopoietic stem and progenitor cells from YS hemogenic endothelium and their decline upon stromal support modulation as intraembryonic organs specialise to assume these functions. The YS therefore functions as three organs in one revealing a multifaceted relay of vital organismal functions as pregnancy proceeds. One Sentence SummaryHuman yolk sac is a key staging post in a relay of vital organismal functions during human pregnancy.

developmental biology↗

bioRxiv2022

Condensin reorganizes centromeric chromatin during mitotic entry into a bipartite structure stabilized by cohesin

The Structural Maintenance of Chromosomes (SMC) complexes cohesin and condensin establish the 3D organization of mitotic chromosomes1-3. Cohesin is essential to maintain sister chromatid pairing until anaphase onset4, while condensin is important for mitotic centromere structure and elastic resistance to spindle forces5-8. Both complexes are also important to form productive kinetochore-spindle attachments6, 8, 9. How condensin and cohesin work together to shape the mitotic centromere to ensure faithful chromosome segregation remains unclear. Here we show by super-resolution imaging, Capture-C analysis and polymer modeling that vertebrate centromeres are partitioned into two distinct condensin-dependent subdomains during mitosis. This bipartite sub-structure is found in human, mouse and chicken centromeres and also in human neocentromeres devoid of satellite repeats, and is therefore a fundamental feature of vertebrate centromere identity. Super-resolution imaging reveals that bipartite centromeres assemble bipartite kinetochores with each subdomain capable of binding a distinct microtubule bundle. Cohesin helps to link the centromere subdomains, limiting their separation in response to mitotic spindle forces. In its absence, separated bipartite kinetochores frequently engage in merotelic spindle attachments. Consistently, uncoupling of centromere subdomains is a common feature of lagging chromosomes in cancer cells. The two-domain structure of vertebrate regional centromeres described here incorporates architectural roles for both condensin and cohesin and may have implications for avoiding chromosomal instability in cancer cells.

cell biology↗

bioRxiv2022

Metabolic, Fibrotic, and Splicing Pathways Are All Altered in Emery-Dreifuss Muscular Dystrophy Spectrum Patients to Differing Degrees

Emery-Dreifuss muscular dystrophy (EDMD) is a genetically and clinically variable disorder. Previous attempts to use gene expression changes find its pathomechanism were unavailing, so we here engaged a functional pathway analysis. RNA-Seq was performed on cells from 10 patients diagnosed with an EDMD spectrum disease with different mutations in 7 genes. Upon comparing to controls, the pathway analysis revealed that multiple genes involved in fibrosis, metabolism, myogenic signaling, and splicing were affected in all patients. Splice variant analysis revealed alterations of muscle-specific variants for several important muscle genes. Deeper analysis of metabolic pathways revealed a reduction in glycolytic and oxidative metabolism and reduced numbers of mitochondria across a larger set of 14 EDMD patients and 7 controls. Intriguingly, the gene expression signatures segregated the patients into three subgroups whose distinctions could potentially relate to differences in clinical presentation. Finally, differential expression analysis of miRNAs changing in the patients similarly highlighted fibrosis, metabolism, and myogenic signaling pathways. This pathway approach revealed a clear EDMD signature that can both be used as the basis for establishing a biomarker panel specific to EDMD and direct further investigation into its pathomechanism. Furthermore, the segregation of specific gene changes into three distinct categories that appear to correlate with clinical presentation may be developed into prognostic biomarkers, though this will first require their testing in a wider set of patients with more clinical information.

genetics↗

bioRxiv2022

Evaluation of CA repeat arrays as targets for MeCP2 function in the brain

The DNA binding protein MeCP2 is reported to bind methylated cytosine in CG and CA motifs in genomic DNA, but it was recently proposed that arrays of tandemly repeated CA containing either methylated or hydroxymethylated cytosine are the primary targets for MeCP2 binding and function. Here we investigated the predictions of this hypothesis using a range of published datasets. We failed to detect enrichment of cytosine modification at genomic CA repeat arrays in mouse brain regions and found no evidence for preferential MeCP2 binding at CA repeats. Moreover, we did not observe a correlation between the CA repeat density near genes and their degree of transcriptional deregulation when MeCP2 was absent. Our results do not provide support for the hypothesis that CA repeats are key mediators of MeCP2 function. Instead, we found that CA repeats are subject to CAC methylation to a degree that is typical of the surrounding genome and contribute modestly to MeCP2-mediated modulation of gene expression in accordance with their content of this canonical target motif.

genomics↗

bioRxiv2021

Systemic influences of mammary cancer on monocytes in mice

There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and blood, particularly the myeloid compartment. Here we show using mouse models of breast cancer caused by the mammary epithelial expression of the Polyoma middle T antigen that there is an increase in the number of circulating and splenic monocytes. In the circulation, cancer does not affect ratios of classical to non-classical populations monocytes nor their halflives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. In the bone marrow cancer does not change monocytic progenitor number is unaffected but the proliferation rate of monocytes is higher thus providing an explanation for expansion in the circulating number. Deep RNA sequencing of these monocytic populations reveals cancer causes changes in the classical monocyte compartment with changes evident in bone marrow monocytes but more in the blood suggesting influences in both compartments. Down regulation of interferon type 1 signalling and antigen presentation were the most prominent. Consistent with this analysis down regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, transcription factors required for type 1 interferon signalling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, mouse models of cancer may be insufficient to study the systemic influences of human cancer.

cancer biology↗

bioRxiv2021

Single cell mRNA signals reveal a distinct developmental state of KMT2A-rearranged infant B-cell acute lymphoblastic leukemia

Infant B-cell acute lymphoblastic leukemia (B-ALL) has not followed the increasing trend towards cure seen in other childhood B-ALLs. The prognosis for infants with KMT2A gene fusions is especially poor, and the origins of this aggressive leukemia remain unknown. Here, we investigated the developmental state of KMT2A-rearranged infant B-ALL within hematopoietic hierarchies of human fetal bone marrow, using bulk mRNA meta-analysis of childhood leukemia and examination of single lymphoblast transcriptomes. KMT2A-rearranged infant B-ALL was uniquely dominated by an early lymphocyte precursor (ELP) state. Direct comparison of infant lymphoblasts with ELP cells distilled the core oncogenic transcriptome of cancer cells which harboured potentially targetable hybrid myeloid-lymphoid features. Overall our quantitative molecular analyses demonstrate a distinct developmental state of KMT2A-rearranged infant B-ALL.

genomics↗

bioRxiv2021

Intrinsic and extrinsic regulation of human fetal bone marrow haematopoiesis and perturbations in Down syndrome

Throughout postnatal life, haematopoiesis in the bone marrow (BM) maintains blood and immune cell production. Haematopoiesis first emerges in human BM at 12 post conception weeks while fetal liver (FL) haematopoiesis is still expanding. Yet, almost nothing is known about how fetal BM evolves to meet the highly specialised needs of the fetus and newborn infant. Here, we detail the development of fetal BM including stroma using single cell RNA-sequencing. We find that the full blood and immune cell repertoire is established in fetal BM in a short time window of 6-7 weeks early in the second trimester. Fetal BM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell (DC) subsets emerging for the first time. B-lymphocyte expansion occurs, in contrast with erythroid predominance in FL at the same gestational age. We identify transcriptional and functional differences that underlie tissue-specific identity and cellular diversification in fetal BM and FL. Finally, we reveal selective disruption of B-lymphocyte, erythroid and myeloid development due to cell intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in the fetal BM from constitutional chromosome anomaly Down syndrome during this crucial developmental time window.

developmental biology↗

bioRxiv2021

Classical macrophage polarisation is limited by human β-defensin 3 via an autocrine IL-4 dependent process.

Human {beta}-defensin 3 (HBD3), is an anti-microbial host-defence peptide, that can rapidly enter macrophages to modulate TLR4 responses to lipopolysaccharide. However, the molecular mechanisms by which HBD3 exerts this anti-inflammatory influence remain unclear. Here, we show mice deleted for the orthologue of HBD3 have an increased acute lipopolysaccharide response in vivo. Furthermore, we found that HBD3 limited the response of macrophages to classical activation, and contemporaneously drove expression of IL-4. An increase in markers of alternative activation, and a change in metabolic flux was also observed. Consistent with these results, HBD3 enhanced the IL-4 mediated polarisation of naive macrophages. Finally, we demonstrate that the ability of HBD3 to limit macrophage classical activation requires IL-4R. These data reveal a previously unrecognised role for HBD3 in influencing the polarisation state of macrophages to enable a state conducive for repair and resolution. SYNOPSIS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/442606v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f81c9aorg.highwire.dtl.DTLVardef@11dca23org.highwire.dtl.DTLVardef@c1e241org.highwire.dtl.DTLVardef@ed46ed_HPS_FORMAT_FIGEXP M_FIG C_FIG The anti-microbial host-defence peptide, Human {beta}-defensin 3 (HBD3), is shown here to modulate the inflammatory response to classical activation by promoting alternative activation through IL-4R, to enable a state conducive for repair and resolution. O_LIKnockout mice for the orthologous gene for HBD3, demonstrate increased acute lipopolysaccharide inflammatory response. C_LIO_LIHBD3 limited the classical activation of macrophages polarised with LPS/IFN{gamma} and drove expression of IL-4. Cells also displayed increase in alternative activation markers and promotion of oxidative phosphorylation. C_LIO_LIHBD3 enhanced the IL-4-mediated activation of naive macrophages. C_LIO_LIThe ability of HBD3 to limit macrophage classical activation and contemporaneously promote alternative activation required IL-4R. C_LI

immunology↗

bioRxiv2021

Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy

Little is known about the proteins that direct the highly conserved patterns of spatial genome organisation in fat. Here we report that adipocyte-specific knockout of the gene encoding nuclear envelope protein Tmem120a disrupts fat genome organisation, thus causing a novel lipodystrophy syndrome. Tmem120a deficiency broadly suppresses lipid metabolism pathway gene expression and induces myogenic gene expression by repositioning genes, enhancers and miRNA-encoding loci between the nuclear periphery and interior. Tmem120a-/- mice, particularly females, exhibit a lipodystrophy syndrome similar to human familial partial lipodystrophy FPLD2, with profound insulin resistance and metabolic defects that manifests upon exposure to an obesogenic diet. Interestingly, similar genome organisation defects occurred in cells from FPLD2 patients that harbour nuclear envelope protein laminA mutations. Our data suggest TMEM120A may mediate/instigate novel categories of adipose tissue dysfunction across the adiposity spectrum and provide a new miRNA-based mechanism possibly driving the unexplained muscle hypertrophy in human lipodystrophy.

cell biology↗

bioRxiv2021

Single-cell atlas of human oral mucosa reveals a stromal-neutrophil axis regulating tissue immunity in health and inflammatory disease

The oral mucosa remains an understudied barrier tissue rich in exposure to antigens, commensals and pathogens. Moreover, it is the tissue where one of the most prevalent human microbe-triggered inflammatory diseases, periodontitis, occurs. To understand this complex environment at the cellular level, we assemble herein a human single-cell transcriptome atlas of oral mucosal tissues in health and periodontitis. Our work reveals transcriptional diversity of stromal and immune cell populations, predicts intercellular communication and uncovers an altered immune responsiveness of stromal cells participating in tissue homeostasis and disease at the gingival mucosa. In health, we define unique populations of CXCL1,2,8-expressing epithelial cells and fibroblasts mediating immune homeostasis primarily through the recruitment of neutrophils. In disease, we further observe stromal, particularly fibroblast hyper-responsiveness linked to recruitment of leukocytes and neutrophil populations. Ultimately, a stromal-neutrophil axis emerges as a key regulator of mucosal immunity. Pursuant to these findings, most Mendelian forms of periodontitis were shown to be linked to genetic mutations in neutrophil and select fibroblast-expressed genes. Moreover, we document previously unappreciated expression of known pattern- and damage-recognition receptors on stromal cell populations in the setting of periodontitis, suggesting avenues for triggering stromal responses. This comprehensive atlas offers an important reference for in-depth understanding of oral mucosal homeostasis and inflammation and reveals unique stromal-immune interactions implicated in tissue immunity.

systems biology↗

bioRxiv2020

Poised cell circuits in human skin are activated in disease

The human skin confers biophysical and immunological protection through a complex cellular network that is established early in development. We profiled ~500,000 single cells using RNA-sequencing from healthy adult and developing skin, and skin from patients with atopic dermatitis and psoriasis. Our findings reveal a predominance of innate lymphoid cells and macrophages in developing skin in contrast to T cells and migratory dendritic cells in adult skin. We demonstrate dual keratinocyte differentiation trajectories and activated cellular circuits comprising vascular endothelial cells mediating immune cell trafficking, disease-specific clonally expanded IL13/IL22 and IL17A/F-expressing lymphocytes, epidermal IL23-expressing dendritic cells and inflammatory keratinocytes in disease. Our findings provide key insights into the dynamic cellular landscape of human skin in health and disease. One Sentence SummarySingle cell atlas of human skin reveals cell circuits which are quantitatively and qualitatively reconfigured in inflammatory skin disease.

immunology↗

bioRxiv2020

Neuronal non-CG methylation is an essential target for MeCP2 function

SUMMARYDNA methylation is implicated in neuronal biology via the protein MeCP2, mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 corepressor complexes to methylated cytosine in the CG dinucleotide, but also to non-CG methylation, which is abundant specifically in neuronal genomes. To test the biological significance of its dual binding specificity, we replaced the MeCP2 DNA binding domain with an orthologous domain whose specificity is restricted to mCG motifs. Knock-in mice expressing the domain-swap protein displayed severe Rett syndrome-like phenotypes, demonstrating that interaction with sites of non-CG methylation, specifically the mCAC trinucleotide, is critical for normal brain function. The results support the notion that the delayed onset of Rett syndrome is due to the late accumulation of both mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2-null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of particular relevance to the Rett syndrome phenotype.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗