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Results for “cell biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2023

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and thereby STAT3 mRNA levels. However, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression.

molecular biology↗

bioRxiv2024

Strategies for Genetic Manipulation of the Halotolerant Black Yeast H. werneckii: Ectopic DNA integration and marker-free CRISPR/Cas9 Trasformation

Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using PEG-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology. ImportanceHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in a research program studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, usage of drugs to target a particular pathway or heterologous expression of its gene in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live cell imaging of cellular processes and reverse genetic approaches.

microbiology↗

bioRxiv2022

CELL-E: Biological zero-shot text-to-image synthesis for protein localization prediction

Accurately predicting cellular activities of proteins based on their primary amino acid sequences would greatly improve our understanding of the proteome. In this paper, we present CELL-E, a text-to-image transformer architecture that generates a 2D probability density map of protein distribution within cells. Given a amino acid sequence and a reference image for cell or nucleus morphology, CELL-E offers a more direct representation of protein localization, as opposed to previous in silico methods that rely on pre-defined, discrete class annotations of protein localization to subcellular compartments.

bioinformatics↗

bioRxiv2021

Quantifying nanotherapeutics penetration using hydrogel based microsystem as a new 3D in-vitro platform

The huge gap between 2D in vitro assays used for drug screening, and the in vivo 3D-physiological environment hampered reliable predictions for the route and accumulation of nanotherapeutics in vivo. For such nanotherapeutics, Multi-Cellular Tumour Spheroids (MCTS) is emerging as a good alternative in vitro model. However, the classical approaches to produce MCTS suffer from low yield, slow process, difficulties in MCTS manipulation and compatibility with high-magnification fluorescent optical microscopy. On the other hand, spheroid-on-chip set-ups developed so far require a microfluidic practical knowledge difficult to transfer to a cell biology laboratory. We present here a simple yet highly flexible 3D-model microsystem consisting of agarose-based microwells. Fully compatible with the multi-well plates format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging including live high-resolution optical microscopy can be performed in-situ, with no manipulation of spheroids. As a proof-of-principle of the relevance of such in vitro platform for nanotherapeutics evaluation, this study investigates the kinetic and localization of nanoparticles within colorectal cancer MCTS cells (HCT-116). The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI (AGuIX(R), currently implicated in clinical trials as effective radiosensitizers for radiotherapy) and confocal microscopy after addition of Cy 5.5. We show that the amount of AGuIX(R) nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX(R) nanoparticles within MCTS. The nanoparticles are first found in both extracellular and intracellular space of MCTS. While the extracellular part is washed away after few days, we evidenced intracellular localisation of AGuIX(R), mainly within lysosomes compartment, but also occasionally within mitochondria. Our agarose-based microsystem appears hence as a promising 3D in vitro user-friendly platform for investigation of nanotherapeutics transport, ahead of in vivo studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/427020v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@bd48adorg.highwire.dtl.DTLVardef@ee7502org.highwire.dtl.DTLVardef@143ee60org.highwire.dtl.DTLVardef@d6f5ca_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

bioengineering↗

bioRxiv2021

Optimized immunoglobulin knock-ins using Cas9 reveal peritoneal B cell lineage relationships in vivo

Immunoglobulin (Ig) knock-in mice are valuable tools in basic and translational immunological research. Here we present "Speed-Ig," a rapid Cas9-based method for generating Ig knock-in mouse lines with high on-target integration rates at both heavy and light chain alleles. With standardized target sites and promoter regions, Speed-Ig mice can be used for comparative studies of B cell biology and vaccine optimization in vivo. We used Speed-Ig to create panels of mice with Ig pairs derived from B-1a, B-1b, and B-2 cells. Surprisingly, B-1b and B-2 Ig pairs drove both B-1b and B-2 phenotypes, suggesting a previously unknown lineage relationship between these subsets. We then confirmed the B-1:B-2 relationship with transcription factor reporter lines and through adoptive cell transfer experiments. In summary, our Ig knock-in approach facilitated the discovery of previously unappreciated aspect of innate-like B cell biology.

immunology↗

bioRxiv2023

Emergent order in epithelial sheets by interplay of cell divisions and cell fate regulation

The fate choices of stem cells between self-renewal and differentiation are often tightly regulated by paracrine (cell-cell) signalling. Here, we assess how the interplay between cell division, differentiation, and short range paracrine signalling can affect the macroscopic ordering of cell types in self-renewing epithelial sheets, by studying a simple spatial cell fate model with cells being arranged on a 2D lattice. We show in this model that if cells commit to their fate directly upon cell division, macroscopic domains of cells of the same type emerge, if at least a small proportion of divisions are symmetric, except if signalling interactions are laterally inhibiting. In contrast, if cells are first licensed to differentiate, yet retaining the possibility to return to their naive state, macroscopic order only emerges if the signalling strength exceeds a critical threshold: if then the signalling interactions are laterally inducing, macroscopic domains emerge as well. Lateral inhibition, on the other hand, can in that case generate macroscopic patterns of alternating cell types (checkerboard pattern), yet only if the proportion of symmetric divisions is sufficiently low. These results can be understood theoretically by an analogy to phase transitions in spin systems known from statistical physics. Author summaryA fundamental question in stem cell biology is how a cells choice to differentiate or not (cell fate choices), is regulated through communication with other cells in a tissue, and whether these choices are a one-way path or to some degree reversible. However, measuring this in living animals is very difficult and often impossible, since this requires to make videos of cells inside the body with a microscope. Here, we employ a simple mathematical model for the fate choices of stem cells when they are regulated by communication with nearby cells in the tissue. We show that different means of cell fate choice and cell communication can lead to qualitatively different patterns of cell types: macroscopic domains, checkerboard patterns, or randomly disordered distributions, depending on the character of cell communication, and whether cell fate is one-way or reversible. Our analysis therefore shows that those aspects of stem cell activity, which are otherwise difficult to measure, can be distinguished by observing macroscopic patterns of cell types.

developmental biology↗

bioRxiv2020

Live cell imaging of the hyperthermophilic archaeon Sulfolobus acidocaldarius identifies complementary roles for two ESCRTIII homologues in ensuring a robust and symmetric cell division

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details.

microbiology↗

bioRxiv2018

Cellular structure analysis based on magnetic induction FEM simulations and measurements

Biological samples exhibit frequency dependent spectra caused by the dispersion mechanism which describes a phenomenon of dielectric relaxation due to the interaction between electromagnetic field and biological samples at cellular levels. Changes in cellular structure such as cellular shape, membrane thickness and integrity could affect bio-impedance spectra.\n\nIn this paper, the influence of cellular shape, membrane thickness and integrity on dielectric properties of biological cells suspension is simulated using a custom developed FEM solver. In its 2D version, the AC conduction case is simulated. In its 3D version, magnetic induction case is simulated. And a new method for calculating the equivalent complex conductivity of biological cell suspension along the eddy current direction is introduced.\n\nMembrane integrity on beta dispersion was experimentally investigated using AC conduction (contact electrode) method on potato samples.\n\nThis suggests that bio-impedance measurements could provide indication of cellular structural changes of biological samples. This could be useful for biomedical, pharmaceutical and food inspection applications.

biophysics↗

bioRxiv2020

Rational design and implementation of a chemically inducible hetero-trimerization system

Chemically inducible dimerization (CID) uses a small molecule to induce binding of two different proteins. CID tools exemplified by the FKBP/FRB/rapamycin system have been widely employed to probe molecular events inside and outside cells. While various CID tools are available, chemically inducible trimerization (CIT) has not been developed, due to inherent challenges in designing or identifying a chemical that simultaneously binds three proteins with high affinity and target specificity. Nevertheless, by introducing a third recruitable component, CIT could enable versatile applications. Here, we devised the CIT by rationally splitting FRB and FKBP. Based on cellular and structural datasets, select split pairs of FRB or FKBP underwent efficient trimerization with full length FKBP or FRB, respectively, upon addition of rapamycin. We also demonstrated its potential for cellular applications by rapidly inducing tri-organellar plasma membrane-ER-mitochondria junctions, and by perturbing intended membrane lipids exclusively at the plasma membrane-ER membrane contact sites. By conferring one additional condition to what is achievable with CID, CIT expands the types of manipulation in single live cells, to address cell biology questions otherwise intractable, and engineer cell functions for future synthetic biology applications.

cell biology↗

bioRxiv2024

The use of thermostable fluorescent proteins for live imaging in Sulfolobus acidocaldarius

Among hyperthermophilic organisms, in vivo protein localization is challenging due to the high growth temperatures that can disrupt proper folding and function of mostly mesophilic-derived fluorescent proteins. While protein localization in the thermophilic model archaeon S. acidocaldarius has been achieved using antibodies with fluorescent probes in fixed cells, the use of thermostable fluorescent proteins in thermophilic archaea has so far been unsuccessful. Given the significance of live protein localization in the field of archaeal cell biology, we aimed to identify fluorescent proteins for use in S. acidocaldarius. To achieve this, we expressed various previously published and optimized thermostable fluorescent proteins along with fusion proteins of interest and analyzed the cells using flow cytometry and (thermo-) fluorescent microscopy. Of the tested proteins, Thermal Green Protein (TGP) exhibited the brightest fluorescence when expressed in Sulfolobus cells. By optimizing the linker between TGP and a protein of interest, we could additionally successfully fuse proteins with minimal loss of fluorescence. TGP-CdvB and TGP-PCNA1 fusions displayed localization patterns consistent with previous immunolocalization experiments. These initial results in protein localization in S. acidocaldarius at high temperatures, combined with recent advancements in thermomicroscopy, open new avenues in the field of archaeal cell biology. This progress finally enables localization experiments in thermophilic archaea, which have so far been limited to mesophilic organisms.

microbiology↗

bioRxiv2019

3D Multicolor Nanoscopy at 10,000 Cells a Day

Diffraction-unlimited single-molecule techniques like STORM and (F)PALM enable three-dimensional (3D) fluorescence imaging at tens of nanometer resolution and are invaluable to investigate sub-cellular organization. The multitude of camera frames required to reconstruct a super-resolved image limits the typical throughput of these techniques to tens of cells per day, rendering these methods incompatible with large-scale cell biological or clinical application. STORM acquisition rates can be increased by over an order of magnitude, however the data volumes of about 40 TB a day and concomitant analysis burdens exceed the capacity of existing workflows. Here we present an integrated platform which transforms SMLM from a trick-pony technique into a work horse for cell biology. We leverage our developments in microscopy-specific data compression, distributed storage, and distributed analysis to automatically perform real-time localization analysis, which enable SMLM at throughputs of 10,000 cells a day. We implemented these advances in a fully-integrated environment that supports a highly-flexible architecture for distributed analysis, enabling quickly- and graphically-reconfigurable analyses to be automatically initiated from the microscope during acquisition, remotely executed, and even feedback and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, the PYthon-Microscopy Environment (PYME), is easily configurable for hardware control on custom microscopes, and includes a plugin framework so users can readily extend all components of their imaging, visualization, and analysis pipeline. PYME is cross-platform, open source, and efficiently puts high-caliber visualization and analysis tools into the hands of both microscope developers and users.

biophysics↗

bioRxiv2022

Heterotrimeric G proteins regulate planarian regeneration and behavior

G protein-coupled receptors (GPCRs) play broad roles in development and stem cell biology, but few roles for GPCR signaling in complex tissue regeneration have been uncovered. Planarian flatworms robustly regenerate all tissues and provide a model with which to explore potential functions for GPCR signaling in somatic regeneration and pluripotent stem cell biology. As a first step toward exploring GPCR function in planarians, we investigated downstream signal transducers that work with GPCRs, called heterotrimeric G proteins. Here, we characterized the complete heterotrimeric G protein complement in Schmidtea mediterranea for the first time and found that seven heterotrimeric G protein subunits promote regeneration. We further characterized two subunits critical for regeneration, Gq1 and G{beta}1-4a, finding that they promote the late phase of anterior polarity re-establishment, likely through anterior pole-produced Follistatin. Incidentally, we also found that five heterotrimeric G proteins modulate planarian behavior. We further identified a putative serotonin receptor, gcr052, that we propose works with G{beta}x2 in planarian locomotion, demonstrating the utility of our strategy for identifying relevant GPCRs. Our work provides foundational insight into roles of heterotrimeric G proteins in planarian biology and serves as a useful springboard towards broadening our understanding of GPCR signaling in adult tissue regeneration.

developmental biology↗

bioRxiv2026

Spatial confinement shapes organelle architecture and remodeling in axons

Axons are extended cellular compartments that mediate neuronal connectivity over extraordinary distances, placing unique demands on local organelle organization and trafficking. How these geometric constraints influence organelle architecture remains poorly understood. Here, we used cryo-electron tomography and quantitative morphometric analyses to define the three-dimensional ultrastructural organization of dorsal root ganglion axons in a near-native state. We identify distinct vesicle populations with tightly versus broadly constrained size distributions and reveal a continuum of endolysosomal and autophagic intermediates that highlight the dynamic nature of membrane remodeling in growing axons. Unexpectedly, we observe vesicular structures enclosed within the lumen of the endoplasmic reticulum, suggesting a previously undescribed mechanism of ER membrane remodeling. Across multiple organelle classes, morphology and size are constrained by axonal geometry. This principle is most evident in mitochondria, which undergo dramatic narrowing and remodeling at varicosity - axon boundaries to traverse confined axonal segments. Together, these findings reveal spatial confinement as a fundamental organizing principle of axonal cell biology. SUMMARYCryo-electron tomography establishes a quantitative framework for organelle organization in axons. Diverse membrane trafficking pathways, including endolysosomal intermediates and mitochondria, exhibit structural adaptations to axonal geometry, identifying spatial confinement as a fundamental organizing principle of axonal cell biology.

neuroscience↗

bioRxiv2023

Machine Learning-based Phenotypic Imaging to Characterise the Targetable Biology of Plasmodium falciparum Male Gametocytes for the Development of Transmission-Blocking Antimalarials

Preventing parasite transmission from humans to mosquitoes is recognised to be critical for achieving elimination and eradication of malaria. Consequently developing new antimalarial drugs with transmission-blocking properties is a priority. Large screening campaigns have identified many new transmission-blocking molecules, however little is known about how they target the mosquito-transmissible Plasmodium falciparum stage V gametocytes, or how they affect their underlying cell biology. To respond to this knowledge gap, we have developed a machine learning image analysis pipeline to characterise and compare the cellular phenotypes generated by transmission-blocking molecules during male gametogenesis. Using this approach, we studied 40 molecules, categorising their activity based upon timing of action and visual effects on the organisation of tubulin and DNA within the cell. Our data both proposes new modes of action and corroborates existing modes of action of identified transmission-blocking molecules. Furthermore, the characterised molecules provide a new armoury of tool compounds to probe gametocyte cell biology and the generated imaging dataset provides a new reference for researchers to correlate molecular target or gene deletion to specific cellular phenotype. Our analysis pipeline is not optimised for a specific organism and could be applied to any fluorescence microscopy dataset containing cells delineated by bounding boxes, and so is extendible to any disease model.

microbiology↗

bioRxiv2021

Global analysis of cell behavior and protein localization dynamics reveals region-specific functions for Shroom3 and N-cadherin during neural tube closure.

Failures of neural tube closure are common and serious birth defects, yet we have a poor understanding of the interaction of genetics and cell biology during neural tube closure. Additionally, mutations that cause neural tube defects (NTDs) tend to affect anterior or posterior regions of the neural tube but rarely both, indicating a regional specificity to NTD genetics. To better understand the regional specificity of cell behaviors during neural tube closure, we analyzed the dynamic localization of actin and N-cadherin via high-resolution tissue-level time-lapse microscopy during Xenopus neural tube closure. To investigate the regionality of gene function, we generated mosaic mutations in shroom3, a key regulator or neural tube closure This approach elucidates new differences between cell behaviors during cranial/anterior and spinal/posterior neural tube closure, provides mechanistic insight into the function of shroom3 and demonstrates the ability of tissue-level imaging and analysis to generate cell-biological mechanistic insights into neural tube closure.

developmental biology↗

bioRxiv2018

Ezrin enrichment on curved membranes requires a specific conformation or interaction with a curvature-sensitive partner

One challenge in current cell biology is to decipher the biophysical mechanisms governing protein enrichment on curved membranes and the resulting membrane deformation. The ERM protein ezrin is abundant and associated with cellular membranes that are flat or with positive or negative curvatures. Using in vitro and cell biology approaches, we assess mechanisms of ezrins enrichment on curved membranes. We evidence that ezrin (ezrinWT) and its phosphomimetic mutant T567D (ezrinTD) do not deform membranes but self-assemble anti-parallelly, zipping adjacent membranes. EzrinTDs specific conformation reduces intermolecular ezrin interactions, allows binding to actin filaments, and promotes ezrin binding to positively curved membranes. While neither ezrinTD nor ezrinWT senses negative membrane curvature alone, we demonstrate that interacting with curvature sensors I-BAR-domain proteins facilitates ezrin enrichment in negatively curved membrane protrusions. Overall, our work reveals new mechanisms, specific conformation or binding to a curvature sensor partner, for targeting curvature insensitive proteins to curved membranes.

biophysics↗

bioRxiv2020

Computational modelling unveils how epiblast remodelling and positioning rely on trophectoderm morphogenesis during mouse implantation

Understanding the processes by which the mammalian embryo implants in the maternal uterus is a long-standing challenge in embryology. New insights into this morphogenetic event could be of great importance in helping, for example, to reduce human infertility. During implantation the blastocyst, composed of epiblast and trophectoderm, undergoes significant remodelling from an oval ball to an egg cylinder. A main feature of this transformation is symmetry breaking and reshaping of the epiblast into a “cup”. Based on previous studies, we hypothesise that this event is the result of mechanical constraints originating from the trophectoderm, which is also significantly transformed during this process. In order to investigate this hypothesis we propose MG#, an original computational model of biomechanics able to reproduce key cell shape changes and tissue level behaviours in silico. With this model, we simulate epiblast and trophectoderm morphogenesis during implantation. First, our results uphold experimental findings that repulsion at the apical surface of the epiblast is sufficient to drive lumenogenesis. Then, we provide new theoretical evidence that trophectoderm morphogenesis indeed dictates the cup shape of the epiblast and fosters its movement towards the uterine tissue. Together, these results offer mechanical insights into mouse implantation and highlight the usefulness of agent-based modelling methods in the study of embryogenesis.Author summary Computational modelling is increasingly used in the context of biological development. Here we propose a novel agent-based model of biological cell and tissue mechanics to investigate important morphological changes during mouse embryo implantation. Our model is able to replicate key biological cell shape changes and tissue-level behaviour. Simulating mouse implantation with this model, we bring theoretical support to previous experimental observations that lumenogenesis in the epiblast is driven by repulsion, and provide theoretical evidence that changes in epiblast shape during implantation are regulated by trophectoderm development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2017

A duplex MIPs-based biological-computational cell lineage discovery platform

Cell lineage analysis aims to uncover the developmental history of an organism back to its cell of origin1. Recently, novel in vivo methods and technologies utilizing genome editing enabled important insights into the cell lineages of animals2-8. In contrast, human cell lineage remains restricted to retrospective approaches, which still lack in resolution and cost-efficient solutions. Here we demonstrate a scalable platform for human cell lineage tracing based on Short Tandem Repeats (STRs) targeted by duplex Molecular Inversion Probes (MIPs). With this platform we accurately reproduced a known lineage of DU145 cell lines cells9 and reconstructed lineages of healthy and metastatic single cells from a melanoma patient. The reconstructed trees matched the anatomical and SNV references while adding further refinements. Our platform allowed to faithfully recapitulate lineages of developmental tissue formation in cells from healthy donors. In summary, our lineage discovery platform can profile informative STR somatic mutations efficiently and we provide a solid, high-resolution lineage reconstruction even in challenging low-mutation-rate healthy single cells.

systems biology↗