Biology subjects

Hansen, J. N.

Publications and source records attributed to Hansen, J. N..

At least 19 recordsLinked to original sources

bioRxiv2026

Reconstruction of septin higher-order nano-size structures in ovarian cancer cells uncover susceptibility to the septin-targeting small molecule UR214-9

In cancer cells, septins assemble into enigmatic higher-order structures of 300-700 nanometers, including long needle-like filaments, thick perinuclear rings, and cytoplasmic bundles or aggregates. The absence of genetic or pharmacological tools to recapitulate these architectures in-vitro has impeded mechanistic studies of their formation, function, and therapeutic targeting. Here, first, determining the overexpression of septin-2 in epithelial ovarian cancer (EOC) and its association with increased mortalities and dependencies, we select SKOV-3 ovarian cancer cells as a tractable model in which septin supramolecular assemblies can be recreated in-vitro and interrogated. This system shows that the forchlorfenuron (FCF) analog UR214-9 remodels septin architecture, converting co-expressed human septin octamers (SEPT2-SEPT6-SEPT7-SEPT9-SEPT9-SEPT7-SEPT6-SEPT2) into large cytoplasmic aggregates. In parallel, transiently expressed SEPT2 is reorganized into septin-rich noodle-like filaments, perinuclear rings, and web-like networks encircling the nucleus upon UR214-9 treatment. Mechanistically, UR214-9 disrupts the incorporation of SEPT2, SEPT7, and SEPT9 into canonical septin hetero-octamers, resulting in assembly-defective or imperfect oligomers that preferentially reorganize into these aberrant higher-order structures. This aggregation likely prevents septin-2 migration during interphase-to-cleavage furrow transition in NRK-49F-SEPT2-EGFP homozygous cells and impacts SKOV-3 cytokinesis, cell proliferation, adhesion and invasion and migration while sparing ceramide transport to the Golgi, preserving ER and cis-Golgi structure. These effects manifested in reduced growth of ovarian, endometrial and breast cancer xenografts without attracting significant off-target engagements per the global transcriptomic analysis of JIMT1 breast cancer and PANC-1 pancreatic cells. UR214-9 treated animals showed observable safety in animals. Thus, a tool to recreate aberrant septin structures and identification of septins as a druggable cytoskeletal target for ovarian, endometrial, breast and pancreatic cancer by perturbing their hetero-octamerization assembly is presented. SignificanceWe provide a method to reconstruct the higher-order septin architecture observed in cancer cells, to study their assembly and functions. Intriguingly, cancer cells tolerate hetero-oligomeric septins lacking specific subunits, suggesting that compositionally deficient oligomers are not efficiently targeted for degradation, unlike unincorporated septin monomers in normal cells. This tolerance may enable accumulation of structurally aberrant septin complexes acquiring long-needles, rings or thick-aggregates in disease cells. We further show that septin oligomerization can be pharmacologically perturbed. By integrating structural, cellular, and energetic readouts using in-silico techniques, we establish a quantitative framework for septin-targeted modulation, generating UR214-9 as a new chemotype that disrupts septin oligomeric assembly via preventing incorporation of SEPT2/7/9, into canonical hetero-octamers, causes defects in cytokinesis, altered cell migration, viability, and remodels septin-actin architectures, ultimately impairing tumor cell growth. Thus, pharmacological targeting of septin assembly represents a tractable strategy to perturb septin-dependent cellular processes in cancer and neurodegenerative diseases with reported septin dysregulation.

cancer biology↗

bioRxiv2026

Generative machine learning unlocks the first proteome-wide image of human cells

The spatial organization of proteins within cells governs virtually all cellular functions, yet current imaging can simultaneously visualize only tens of proteins, orders of magnitude below the thousands populating a single human cell. Here we present ProtiCelli, a deep generative model that simulates microscopy images for 12,800 human proteins from just three cellular landmark stains. Trained on 1.23 million Human Protein Atlas images, ProtiCelli outperforms existing methods in reconstruction accuracy and textural fidelity, and generalizes to unseen cell types and drug perturbations. Simulated images preserve hierarchical subcellular organization, recapitulate known protein protein interaction landscapes, and resolve compartment-specific functions of moonlighting proteins at single cell resolution. Remarkably, the model infers drug-induced changes in protein expression and localization from cell morphology alone, predicts cell cycle stage without dedicated markers, and enables unsupervised segmentation of subcellular compartments and spatial decomposition of gene sets into functional regions. We leverage ProtiCelli to generate Proteome2Cell, a dataset of 30.7 million simulated images spanning 2,400 virtual cells across 12 human cell lines, enabling hierarchical single-cell models that distinguish conserved from dynamic protein architectures. Integrated into the Human Protein Atlas, Proteome2Cell democratizes exploration of these virtual cells. By computationally bridging the experimental scalability gap, ProtiCelli establishes a foundation for spatial virtual cell modeling.

cell biology↗

bioRxiv2026

A high-resolution spatial map of cilia-associated proteins in the human fallopian tube

Molecular alterations in the fallopian tubes play a pivotal role in the development of cancer and reproductive disorders, yet their molecular landscape at the protein level remains poorly defined. Here, we map key fallopian tube proteins at single-cell resolution utilizing an integrated transcriptomics and proteomics approach. Based on RNA-seq analysis we identify 310 genes with elevated expression in fallopian tube, the majority of which are associated with motile cilia function. We spatially characterize 133 of the corresponding proteins in fallopian tube and other human tissues with motile cilia to subcellular structures of ciliated cells, validating the findings with single-cell RNA-seq and mass-spectrometry data. Eleven proteins previously only studied on the transcript level without information in cilia databases are further analyzed in a hydrosalpinx patient, showing a thinner epithelium, lower density of FOXJ1 expression, and reduced expression of FHAD1, RIIAD1, and C2orf81. Our high-resolution spatial map aids in dissecting the pathways underlaying infertility and diseases linked to cilia-specific functions.

molecular biology↗

bioRxiv2025

Cell shapes decode molecular phenotypes in image-basedspatial proteomics

The diversity of cellular and tissue structures can arise from a few basic cell shapes, which undergo various transformations based on biophysical constraints on cytoskeletal organization. While cellular geometry has been linked with selected biological processes such as polarity, signaling or morphogenesis, the orchestration of the whole proteome in association to cell shape is still poorly understood. In this study, using more than 1 million images of single cells stained for 11,998 proteins across 10 cell lines in the Human Protein Atlas database, we performed an integrated analysis of organelle, pathway and single protein levels in association to a 2D cellular shapespace. We found that cell and nuclear shapes across cell lines exist in a shared continuum. We also found that the subcellular organelle topology varies across cell lines, but remains robust within each cell lines shapespace. At the single protein level, we found that cells of different shapes in the same cell cycle phase might be preparing for different fates, and that many non-cell cycle proteins expressed shape-based abundance variation. Using the same coordinate framework defined by shape, we could analyze the distribution shift of protein spatial localization under drug perturbation.

bioengineering↗

bioRxiv2025

Proximity proteomics of primary cilia in human hypothalamic neurons

Primary cilia are hair-like sensory organelles that project from the cell bodies of most cell types, including appetite-regulatory hypothalamic neurons where they likely help sense metabolic factors to regulate food intake. We hypothesized that characterising the proteins present in the primary cilia of hypothalamic neurons would shed mechanistic insights into their sensory role and identify new therapeutic targets for obesity. We therefore targeted the ascorbate peroxidase APEX2 to primary cilia in human induced pluripotent stem cells (hiPSC)-derived hypothalamic neurons to biotinylate and identify ciliary proteins. Among the cilia-enriched proteins, we identified synaptic proteins, neurotransmitter receptors, and cell-cell adhesion and axon guidance proteins, extending recent findings that primary cilia interact with neuronal synapses. We also found genes associated with increased body weight and metabolic phenotypes that could represent new therapeutic targets including the lysophosphatidic receptor 1 (LPAR1), which we validated is cilia-localized and we confirmed that its ligand (LPA) mediates ciliary shortening. These findings provide insights into the molecular mechanisms by which primary cilia functionally impact appetite-regulatory neurons.

cell biology↗

bioRxiv2025

A high-resolution subcellular map of proteins in ciliated cells

Motile cilia are complex structures regulated by thousands of genes, essential for various physiological functions like respiration and reproduction. Their dysfunction can result in severe conditions like primary ciliary dyskinesia (PCD), highlighting the need for a deeper molecular understanding of their specific ciliary compartments. Interestingly, ciliated cells harbor multiple proteins with limited evidence on biological function, as defined by Functional Evidence (FE) scores, a grading system developed by the Human Proteome Project (HPP). Building upon the stringent antibody validation pipeline of the Human Protein Atlas (HPA) project, we developed a high-throughput workflow that combines a novel multiplex immunohistochemistry protocol with image analysis to investigate protein expression and subcellular localization in motile ciliated cells across five human tissues: nasopharynx, bronchus, fallopian tube, endometrium, and cervix. We spatially mapped >180 proteins, out of which 73% have FE scores 2-5, suggesting that further evidence is needed to establish these proteins biological function. Notably, expression patterns varied between tissues, suggesting that motile cilia proteins are not universally expressed across the different epithelia. Our pipeline constitutes a promising resource for comprehensive mapping of the motile cilia proteome, and a first step towards identifying cilia proteins for functional studies to understand the molecular mechanisms underlying ciliopathies.

cell biology↗

bioRxiv2024

SubCell: Vision foundation models for microscopycapture single-cell biology

Cell morphology and subcellular protein organization provide important insights into cellular function and behavior. These cellular features can be studied using large-scale fluorescence microscopy, and machine learning has become a powerful tool to interpret the resulting images for biological insights. Here, we introduce SubCell, a deep learning model for fluorescence microscopy designed to accurately capture cellular morphology, protein localization, cellular forganization, and biological function beyond what humans can readily perceive. SubCell was trained on the proteome-wide image collection from the Human Protein Atlas with a novel proteome-aware learning objective. SubCell outperforms state-of-the-art methods across a variety of tasks relevant to single-cell biology and generalizes to other fluorescence microscopy datasets without any fine-tuning. Additionally, we construct the first proteome-wide hierarchical map of proteome organization that is directly learned from image data. This vision-based multiscale cell map defines cellular subsystems down to protein complex resolution, reveals proteins with similar functions, and distinguishes dynamic and stable behaviors within cellular compartments. Finally, combining SubCell with a protein sequence model enables a rich multimodal approach to capture gene function better than either vision-only or sequence-only models alone. In conclusion, SubCell creates deep, image-driven representations of cellular architecture that are applicable across diverse biological contexts and datasets.

cell biology↗

bioRxiv2024

A PERTURBATION CELL ATLAS OF HUMAN INDUCED PLURIPOTENT STEM CELLS

Towards comprehensively investigating the genotype-phenotype relationships governing the human pluripotent stem cell state, we generated an expressed genome-scale CRISPRi Perturbation Cell Atlas in KOLF2.1J human induced pluripotent stem cells (hiPSCs) mapping transcriptional and fitness phenotypes associated with 11,739 targeted genes. Using the transcriptional phenotypes, we created a minimum distortion embedding map of the pluripotent state, demonstrating rich recapitulation of protein complexes, such as strong co-clustering of MRPL, BAF, SAGA, and Ragulator family members. Additionally, we uncovered transcriptional regulators that are uncoupled from cell fitness, discovering potential novel pluripotency (JOSD1, RNF7) and metabolic factors (ZBTB41). We validated these findings via phenotypic, protein-interaction, and metabolic tracing assays. Finally, we propose a contrastive human-cell engineering framework (CHEF), a machine learning architecture that learns from perturbation cell atlases to predict perturbation recipes that achieve desired transcriptional states. Taken together, our study presents a comprehensive resource for interrogating the regulatory networks governing pluripotency.

bioengineering↗

bioRxiv2024

AI-readiness for Biomedical Data: Bridge2AI Recommendations

Biomedical research is rapidly adopting artificial intelligence (AI). Yet the inherent complexity of biomedical data preparation requires implementing actionable, robust criteria for ethical and explainable AI (XAI) at the "pre-model" stage, encompassing data acquisition, detailed transformations, and ethical governance. Simple conformance to FAIR (Findable, Accessible, Interoperable, Reusable) Principles is insufficient. Here, we define criteria and practices for reliable AI-readiness of biomedical data, developed by the NIH Bridge to Artificial Intelligence (Bridge2AI) Standards Working Group across seven core dimensions of dataset AI-readiness: FAIRness, Provenance, Characterization, Ethics, Pre-model Explainability, Sustainability, and Computability. Conformance to these criteria provides a basis for pre-model scientific rigor and ethical integrity, mitigating downstream risks of bias and error prior to AI modeling. We apply and evaluate these standards across all four Bridge2AI flagship datasets, spanning functional genomics to clinical medicine, and encode them in machine-actionable metadata bound to the datasets. This framework sets a benchmark for preparing ethical, reusable datasets in biomedical AI and provides standardized methods for reliable pre-model data evaluation.

bioinformatics↗

bioRxiv2024

Cell Maps for Artificial Intelligence: AI-Ready Maps of Human Cell Architecture from Disease-Relevant Cell Lines

This article describes the Cell Maps for Artificial Intelligence (CM4AI) project and its goals, methods, standards, current datasets, software tools, status, and future directions. CM4AI is the Functional Genomics Data Generation Project in the U.S. National Institute of Healths (NIH) Bridge2AI program. Its overarching mission is to produce ethical, AI-ready datasets of cell architecture, inferred from multimodal data collected for human cell lines, to enable transformative biomedical AI research.

systems biology↗

bioRxiv2024

Spatial Dynamics of the Developing Human Heart

Heart development relies on a topologically defined interplay between a diverse array of cardiac cells. We finely curated spatial and single-cell measurements with subcellular imaging-based transcriptomics validation to explore spatial dynamics during early human cardiogenesis. Analyzing almost 80,000 individual cells and 70,000 spatially barcoded tissue regions between the 5.5th and 14th postconceptional weeks, we identified 31 coarse- and 72 fine-grained cell states and mapped them to highly resolved cardiac cellular niches. We provide novel insight into the development of the cardiac pacemaker-conduction system, heart valves, and atrial septum, and decipher heterogeneity of the hitherto elusive cardiac fibroblast population. Furthermore, we describe the formation of cardiac autonomic innervation and present the first spatial account of chromaffin cells in the fetal human heart. We support independent exploration of our datasets by an open-access, spatially centric interactive viewer. In summary, our study delineates the cellular and molecular landscape of the developing hearts architecture, offering links to genetic causes of heart disease.

developmental biology↗

bioRxiv2024

Opposing roles of resident and infiltrating immune cells in the defence against Legionella longbeachae via IL-18R/IFN-γ/ROS axis

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires pneumonia, is poorly understood. Here we investigated the specific roles of tissue-resident alveolar macrophages (AM) and infiltrating phagocytes during infection with this pathogen. AM were the predominant cell type that phagocytosed bacteria o day after infection. Three and five days after infection, AM numbers were greatly reduced while there was an influx of neutrophils and later monocyte-derived cells (MC) into lung tissue. AM carried greater numbers of viable L. longbeachae than neutrophils and MC, which correlated with a higher capacity of L. longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection whereas neutrophils and MC were required for efficient bacterial clearance. IL-18 was important for IFN-{gamma} production by IL-18R+ NK cells and T cells which, in turn, stimulated ROS-mediated bactericidal activity in neutrophils resulting in restriction of L. longbeachae infection. Ciliated epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L. longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L. longbeachae infection that may be manipulated to improve protective responses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/578217v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@107c421org.highwire.dtl.DTLVardef@dd76f5org.highwire.dtl.DTLVardef@1acf746org.highwire.dtl.DTLVardef@9e2c17_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAM serve as a replicative niche and promote L. longbeachae infection. C_LIO_LIInfiltrating neutrophils and MC kill L. longbeachae. C_LIO_LIIFN-{gamma} from IL-18R+ NK and T cells stimulates ROS and bacterial clearance. C_LI

immunology↗

bioRxiv2024

High-parametric protein maps reveal the spatial organization in early-developing human lung

The respiratory system, including the lungs, is essential for terrestrial life. While recent research has advanced our understanding of lung development, much still relies on animal models and transcriptome analyses. In this study conducted within the Human Developmental Cell Atlas (HDCA) initiative, we describe the protein-level spatiotemporal organization of the lung during the first trimester of human gestation. Using high-parametric tissue imaging with a 30-plex antibody panel, we analyzed human lung samples from 6 to 13 post-conception weeks, generating data from over 2 million cells across five developmental timepoints. We present a resource detailing spatially resolved cell type composition of the developing human lung, including proliferative states, immune cell patterns, spatial arrangement traits, and their temporal evolution. This represents an extensive single-cell resolved protein-level examination of the developing human lungs and provides a valuable resource for further research into the developmental roots of human respiratory health and disease.

developmental biology↗

bioRxiv2022

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene-editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/y Cylc2+/-, and Cylc1-/y Cylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

developmental biology↗

bioRxiv2022

AdipoQ - a simple, open-source software to quantify adipocyte morphology and function in tissues and in vitro

The different adipose tissues can be distinguished according to their function. For example, white adipose tissue (WAT) stores energy in form of lipids, whereas brown adipose tissue (BAT) dissipates energy in the form of heat. These functional differences are represented in the respective adipocyte morphology: whereas white adipocytes contain large, unilocular lipid droplets, brown adipocytes contain smaller, multilocular lipid droplets. However, an automated, image-analysis pipeline to comprehensively analyze adipocytes in vitro in cell culture as well as ex vivo in tissue sections is missing. We here present AdipoQ, an open-source software implemented as ImageJ plugins that allows to analyze adipocytes in tissue sections and in vitro after histological and/or immunofluorescent labelling. AdipoQ is compatible with different imaging modalities and staining methods, allows batch processing of large datasets and simple post-hoc analysis, provides a broad band of parameters, and allows combining multiple fluorescent read-outs. Thereby, AdipoQ is of immediate use not only for basic research but also for clinical diagnosis.

cell biology↗

bioRxiv2021

Locally synchronized ciliary domains and tissue-scale cilia alignment underlie global metachronal wave patterns

Motile cilia are hair-like cell extensions present in multiple organs of the body. How cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine here experiments, novel analysis tools, and theory to address this knowledge gap. We investigate collective dynamics of cilia in the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Despite the fact that synchronization is local only, we observed global patterns of traveling metachronal waves across the multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right nose, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment are sufficient to generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping.

biophysics↗

bioRxiv2021

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

Soluble adenylyl cyclase (sAC: ADCY10) is essential for activating dormant sperm. Studies of freshly dissected mouse sperm identified sAC as needed for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in human sperm. Unlike dissected mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. Even in ejaculated human sperm, TDI-10229 interrupts stimulated motility and capacitation, and it prevents acrosome reaction in capacitated sperm. At present, there are no non-hormonal, pharmacological methods for contraception. Because sAC activity is required post-ejaculation at multiple points during the sperms journey to fertilize the oocyte, sAC inhibitors define candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in females.

pharmacology and toxicology↗

bioRxiv2021

Diversity and Function of Motile Ciliated Cell Types within Ependymal Lineages of the Zebrafish Brain

Motile cilia defects impair cerebrospinal fluid (CSF) flow, and can cause brain and spine disorders. To date, the development of ciliated cells, their impact on CSF flow and their function in brain and axial morphogenesis are not fully understood. Here, we have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricular surface undergoes significant restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs are translationally polarized, co-exist with monociliated cells and generate directional flow patterns. Moreover, these ciliated cells have different developmental origins, and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from specific brain regions or global perturbation of multiciliation does not affect overall brain or spine morphogenesis, but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.

neuroscience↗