Biology subjects

Wrana, J. L.

Publications and source records attributed to Wrana, J. L..

13 recordsLinked to original sources

bioRxiv2026

Spatiotemporal remodeling of cytoskeletal and junction networks during somatic cell reprogramming

Summary/AbstractReprogramming somatic cells into induced pluripotent stem cells involves a dramatic reorganization of the cytoskeleton and junctions during the critical mesenchymal-to-epithelial transition stage. While protein abundance changes have been profiled, the spatiotemporal dynamics of protein-protein associations involving these structural components remain poorly resolved. Here, we present a time-resolved proximity proteomics resource that maps cytoskeletal and junctional remodeling across 27 baits during the early stages of reprogramming. We identified over 1100 high-confidence interactions, including many not previously reported, capturing the dynamic reorganization of cell architecture. By integrating proximity-dependent biotinylation with quantitative proteomics, we distinguished spatial relocalizations from abundance-driven effects. Dynamic redistributions of actin regulators and desmosomal proteins were observed, and a targeted short interfering RNA screen uncovered early acting structural proteins essential for colony formation. Our findings reveal adhesion and cytoskeletal maturation as structural bottlenecks in reprogramming and provide a broadly applicable framework for mapping subcellular remodeling during dynamic cell fate transitions.

systems biology↗

bioRxiv2026

Physiological perfusion of human vasculature reveals a YAP/TAZ-Apelin switch linking intraluminal flow to endothelial state transitions and vessel remodeling

Vascular flow delivers nutrients and imposes hemodynamic forces that govern vessel behavior in health and disease, yet fully human systems that recapitulate and tune physiological intraluminal flow in three-dimensional (3D) tissues are lacking. We developed VIVOS (Vascularized In Vitro Organ Systems), a platform that couples perfused human vascular beds to tunable pumps, generating continuous intraluminal flow through millimetre-scale vessels and 3D tissues at physiological shear stresses and pressures. VIVOS supports integration and perfusion of diverse human organoids and tissues, including lung organoids, cerebral organoids, vascular organoids, breast spheroids, and human retinal explants, as well as enables direct measurement and control of pressure, shear stress, and perfusion-dominant compound transport over extended culture periods. By tuning intraluminal flow and applying single-cell transcriptomics, we uncover a remodeling program in which laminar shear stress acts through a YAP/TAZ-TEAD "switch" to rewire an Apelin ligand-receptor axis and bias tip-stalk endothelial states, reshaping human vascular networks and linking hemodynamic cues to cell state transitions. We further model fast-flow arteriovenous malformations (AVMs) from Hereditary Hemorrhagic Telangiectasia and show that BMP9 constrains vessel caliber and perfusion while antagonizing a VEGF-driven angiogenic program, generating flow-quantified AVM-like lesions in a fully human 3D context. Together, these findings establish VIVOS as a generalizable platform that links physiological intraluminal flow to endothelial state transitions and vessel remodeling, enabling preclinical testing and mechanistic dissection of flow-regulated vascular pathologies in perfused 3D human tissues under defined hemodynamic conditions.

bioengineering↗

bioRxiv2024

LINE1 promotes nuclear compartmentalization to repress the 8-cell state in embryonic stem cells

The family of LINE1 transposable elements underwent a massive expansion in mammalian genomes. While traditionally viewed as a mutagenic selfish element, recent studies point to roles for LINE1 in early mouse development, T cell quiescence and neurogenesis. Here we show that human LINE1 RNA is essential for self-renewal and identity of human embryonic stem cells (hESCs). Silencing of LINE1 using either antisense oligonucleotides or CRISPR interference in naive hESCs leads to a strong induction of 8C-like cells (8CLCs). We found that genes derepressed upon LINE1 KD are not uniformly distributed across the genome, with an enrichment for chromosome 19, which includes key markers of the 8C state such as TPRX1. Silencing of TPRX1, but not other putative 8C regulators p53 or H3.XY, suppresses the induction of the 8C program in LINE1 KD hESCs. We found that LINE1 RNA is preferentially localized to the lamina and periphery of the nucleolus in hESCs. Sequencing of Lamina-Associated Domains (LADs) and Nucleolus-Associated Domains (NADs) reveals a preferential association of chromosome 19 with NADs in hESCs. However, 8CLCs have a distinct nucleolar morphology and a lower association of chromosome 19 and TPRX1 loci with the nucleolus relative to naive and primed hESCs, suggesting a role for nucleolar dynamics in the 8CLC-hESC transition. In agreement, LINE1 KD leads to disruption of nucleolar architecture with signs of nucleolar stress. Independent perturbations of the nucleolus induce the 8C program in hESCs. Genes induced by LINE1 KD are enriched for targets of Polycomb Repressive Complex (PRC2), and inhibition of PRC2 leads to a strong induction of 8C genes. Our results indicate that LINE1 coordinates nuclear compartmentalization and chromatin-mediated gene repression to prevent developmental reversion of hESCs. HighlightsO_LIKnockdown of LINE1 induces TPRX1-dependent emergence of 8C-like cells in hESCs. C_LIO_LIGenes de-repressed upon LINE1 KD are enriched for Chr 19 and PRC2 targets. C_LIO_LI8CLCs display dissociation of Chromosome 19 from the nucleolus. C_LIO_LIDisruption of the nucleolus or inhibition of PRC2 strongly induce the 8C program. C_LI

developmental biology↗

bioRxiv2024

PIEZO-dependent mechano-sensing of the niche is essential for intestinal stem cell fate decision and maintenance

Stem cells continuously perceive and respond to various environmental signals to maintain homeostasis. In addition to biochemical factors, the stem cell niche is subjected to mechanical and physical cues. However, it remains unclear how stem cells can sense mechanical signals from their niche in vivo. Since intestinal stem cells constantly and directly face the external environment, we investigated the roles of mechano-sensing PIEZO ion channels in the gut stem cell niche. By employing mouse genetics and performing single-cell RNAseq analysis, we revealed the absolute requirement for PIEZO channels in intestinal stem cell (ISC) state dynamics and maintenance. In vivo measurement of basement membrane region stiffness demonstrated that ISCs reside in a more rigid microenvironment at the bottom of the crypt. Using 3D and 2D organoid systems combined with bioengineered substrates and a cell stretching device, we found that PIEZO channels are activated by high extracellular matrix stiffness and tissue tension to modulate ISC behavior. This study delineates the mechanistic cascade of PIEZO channel activation in ISCs from the upstream extracellular stimuli through the downstream signaling activation that coordinates stem cell fate decision and maintenance.

cell biology↗

bioRxiv2024

Early Inflammation and Interferon Signaling Direct Enhanced Intestinal Crypt Regeneration after Proton FLASH Radiotherapy

Ultra-high dose rate ("FLASH") radiotherapy ([≥]40-60 Gy/s) is a promising new radiation modality currently in human clinical trials. Previous studies showed that FLASH proton radiotherapy (FR) improves toxicity of normal tissues compared to standard proton radiotherapy (SR) without compromising anti-tumor effects. Understanding this normal tissue sparing effect may offer insight into how toxicities from cancer therapy can be improved. Here, we show that compared to SR, FR resulted in improved acute weight recovery and survival in mice after whole-abdomen irradiation. Improved morbidity and mortality after FR were associated with greater proliferation of damage-induced epithelial progenitor cells followed by improved tissue regeneration. FR led to the accelerated differentiation of revival stem cells (revSCs), a rare damage-induced stem cell required for intestinal regeneration, and to qualitative and quantitative changes in activity of signaling pathways important for revSC differentiation and epithelial regeneration. Specifically, FR resulted in greater infiltration of macrophages producing TGF-{beta}, a cytokine important for revSC induction, that was coupled to augmented TGF-{beta} signaling in revSCs. In pericryptal fibroblasts, FR resulted in greater type I IFN (IFN-I) signaling, which directly stimulates production of FGF growth factors supporting revSC proliferation. Accordingly, the ability of FR to improve epithelial regeneration and morbidity was dependent on IFN-I signaling. In the context of SR, however, IFN-I had a detrimental effect and promoted toxicity. Thus, a tissue-level signaling network coordinated by differences in IFN-I signaling and involving stromal cells, immune cells, and revSCs underlies the ability of FLASH to improve normal tissue toxicity without compromising anti-tumor efficacy. One Sentence SummaryFLASH radiation improves normal tissue toxicity without compromising anti-tumor efficacy through a stroma, immune, and revSC signaling network coordinated by IFN-I.

cell biology↗

bioRxiv2024

Bacterial ADP-heptose initiates a revival stem cell program in the intestinal epithelium

The intestinal epithelium has an exceptional capacity to repair following injury, and recent evidence has suggested that YAP-dependent signaling was crucial for the expansion of Clu+ revival stem cells (revSCs) with fetal-like characteristics, which are essential for epithelial regeneration. However, neither the mechanism underlying where these revSCs emerge from nor the nature of the physiological cues that induce this revSC program, are clearly identified. Here, we first demonstrate that Alpk1 and Tifa, which encode the proteins essential for the detection of the bacterial metabolite ADP-heptose (ADP-Hep), were expressed by the stem cell pool in the intestinal epithelium. Treatment of intestinal organoids with ADP-Hep not only induced acute NF-{kappa}B pro-inflammatory signaling but also TNF-dependent apoptosis within the crypt, causing blunted proliferation and acute disruption of the crypt architecture, while also triggering induction of a revSC program. To identify the molecular underpinnings of this process, we performed single-cell RNA-seq analysis of ADP-Hep-treated organoids as well as lineage-tracing experiments. Our data reveal that ADP-Hep induced the specific ablation of the homeostatic intestinal stem cell (ISC) pool. Removal of ADP-Hep resulted in the rapid recovery of ISCs through dedifferentiation of Paneth cells, which transiently acquired revSC features and expressed nuclear YAP. Moreover, lineage tracing from Lyz1+ Paneth cells showed that ADP-Hep triggered Paneth cell de-differentiation towards pluripotent and proliferative cells in organoids. In vivo, revSC emergence in response to irradiation-induced injury was severely blunted in Tifa-deficient mice, suggesting that efficient epithelial regeneration in this model required detection of microbiota-derived ADP-Hep by the ALPK1-TIFA pathway. Together, our work reveals that Paneth cells can serve as the cell of origin for revSC induction in the physiological context of microbial stimulation, and that the transient loss of Alpk1-expressing ISCs is the initiating event for this regenerative process.

cell biology↗

bioRxiv2023

A Comparative Analysis of Imaging-Based Spatial Transcriptomics Platforms

Spatial transcriptomics is a rapidly evolving field, overwhelmed by a multitude of technologies. This study aims to offer a comparative analysis of datasets generated from leading in situ imaging platforms. We have generated spatial transcriptomics data from serial sections of prostate adenocarcinoma using the 10x Genomics Xenium and NanoString CosMx SMI platforms. Additionally, orthogonal single-nucleus RNA sequencing (snRNA-seq) was performed on the same FFPE tissue to establish a reference for the tumors transcriptional profiles. We assessed various technical aspects, such as reproducibility, sensitivity, dynamic range, cell segmentation, cell type annotation, and congruence with single-cell profiling. The practicality of assessing cellular organization and biomarker localization was evaluated. Although fewer genes are measured (CosMx: 960, Xenium: 377, with an overlap of 125), Xenium consistently demonstrates higher sensitivity, a broader dynamic range, and better alignment with single-cell reference profiles. Conversely, CosMxs out-of-the-box segmentation outperformed Xeniums, resulting in noticeable transcript misassignment in Xenium within certain tissue areas. However, the impact of this on the cells transcriptional profile was minimal. Together, this comprehensive comparison of two leading commercial platforms for spatial transcriptomics provides essential metrics for assessing their performance, offering invaluable insights for future research and technological advancements in this dynamic field.

genomics↗

bioRxiv2023

Exosomes promote axon outgrowth and a polarized neuronal morphology by engaging the Wnt-Planar Cell Polarity pathway

In neurons, the acquisition of a polarized morphology is achieved upon the outgrowth of a single axon from one of several neurites. Exosomes or small extracellular vesicles (sEVs) from diverse sources are known to promote the neurite outgrowth and thus may have therapeutic potential. However, the effect of fibroblast-derived exosomes on axon elongation in neurons of the central nervous system under growth permissive conditions remains unclear. Here, we show that fibroblast-derived sEVs promote axon outgrowth and a polarized neuronal morphology in mouse primary embryonic cortical neurons. Mechanistically, we demonstrate that the sEV-induced increase in axon outgrowth requires endogenous Wnts and core PCP components including Prickle, Vangl, Frizzled and Dishevelled. We demonstrate that sEVs are internalized by neurons, colocalize with Wnt7b and induce relocalization of Vangl2 to the distal axon during axon outgrowth. In contrast, sEVs derived from neurons or astrocytes do not promote axon outgrowth, while sEVs from activated astrocytes inhibit elongation. Thus, our data reveals that fibroblast-derived sEVs promote axon elongation through the Wnt-PCP pathway in a manner that is dependent on endogenous Wnts.

neuroscience↗

bioRxiv2023

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming. One-Sentence SummaryAfter severe radiation injury to the intestine, transient p53 activity induces revival stem cells to promote regeneration.

cell biology↗

bioRxiv2023

Integrated single-cell analysis defines the epigenetic basis of castration-resistant prostate luminal cells

Understanding prostate response to castration and androgen receptor signaling inhibitors (ARSI) is critical to improving long-term prostate cancer (PCa) patient survival. Here we use a multi-omics approach on 229,794 single cells to create a mouse single-cell reference atlas better suited to interpreting mouse prostate biology and castration response. Our reference atlas refines single-cell annotations and provides chromatin context, which, when coupled with mouse lineage tracing demonstrates that the castration-resistant luminal cells are distinct from the pre-existent urethra- proximal stem/progenitor cells. Molecular pathway analysis and therapeutic studies further implicate JUN/FOS, WNT/{beta}-Catenin, FOXQ1, NF{kappa}B, and JAK/STAT pathways as the major drivers of castration- resistant luminal populations with high relevance to human PCa. Importantly, we demonstrate the utility of our datasets, which can be explored through an interactive portal (https://visportal.roswellpark.org/data/tang/), to aid in developing novel combination treatments with ARSI for advanced PCa patients. O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/530998v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@8bd384org.highwire.dtl.DTLVardef@1ebc004org.highwire.dtl.DTLVardef@8b4dbforg.highwire.dtl.DTLVardef@19d7052_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants

The omicron variant of concern (VOC) of SARS-CoV-2 was first reported in November 2021 in Botswana and South Africa. Omicron has evolved multiple mutations within the spike protein and the receptor binding domain (RBD), raising concerns of increased antibody evasion. Here, we isolated infectious omicron from a clinical specimen obtained in Canada. The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed. Convalescent sera from unvaccinated individuals infected by the ancestral virus during the first wave of COVID-19 in Canada (July, 2020) demonstrated reduced neutralization against beta and omicron VOCs. Convalescent sera from unvaccinated individuals infected by the delta variant (May-June, 2021) neutralized omicron to significantly lower levels compared to the delta variant. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of the omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Importantly, infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. This data will inform current booster vaccination strategies, and we highlight the need for additional studies to identify longevity of immunity against SARS-CoV-2 and optimal neutralizing antibody levels that are necessary to prevent infection and/or severe COVID-19.

immunology↗

bioRxiv2020

Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells

Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.

neuroscience↗

bioRxiv2020

Comparison of SARS-CoV-2 Indirect and Direct Detection Methods

The COVID-19 pandemic caused by the SARS-CoV-2 virus has placed extensive strain on RNA isolation and RT-qPCR reagents. Rapid development of new test kits has helped to alleviate these shortages. However, comparisons of these new detection systems are largely lacking. Here, we compare indirect methods that require RNA extraction, and direct RT-qPCR on patient samples. For RNA isolation we compared four different companies (Qiagen, Invitrogen, BGI and Norgen Biotek). For detection we compared two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system proved most sensitive, generating comparable results to clinical diagnostic data, and identifying samples ranging from 65 copies - 2.1x105 copies of viral Orf1ab/l. However, the BGI detection system is [~]4x more expensive than other options tested here. With direct RT-qPCR we found that simply adding RNase inhibitor greatly improved sensitivity, without need for any other treatments (e.g. lysis buffers or boiling). The best direct methods were [~]10 fold less sensitive than indirect methods, but reduce sample handling, as well as assay time and cost. These studies will help guide the selection of COVID-19 detection systems and provide a framework for the comparison of additional systems.

microbiology↗