Biology subjects

Mishra, S.

Publications and source records attributed to Mishra, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

A PKM2-YAP reciprocal repartitioning modulates invasion of breast cancer cells

Invasive cancer cells exhibit distinct morphomigrational and metabolic traits when confronted with biophysically variant matrix microenvironments enroute metastasis. Whether dynamical shifts in such traits are interlinked through a common molecular program remains ill-understood. Using triple negative breast cancer cell lines on Collagen I substrata coated hydrogels recapitulating stiffness values of non-cancerous breast tissue and the desmoplasia of tumors, we observed greater cell shape polarization and migration in the latter. Associated lower lactate and pyruvate levels in such conditions motivated us to examine their pyruvate kinase M2 expression, which showed nuclear and cytoplasmic localization in softer and stiffer environments respectively. Pharmacologically impairing PKM2 activity in stiffer substrata decreased migration and shape polarization of cancer cells while increasing lactate and pyruvate levels. In contrast, increasing its activity on softer substrata attenuated cancer cell migration and elongation. We assayed for localization of the mechanosensory protein YAP upon PKM2 activity modulation: PKM2 activation increased nuclear YAP localization on soft substrata. Pharmacologically inhibiting YAP on stiff substrata not just decreased migration but also increased nuclear localization of PKM2 and lactate and pyruvate levels. We propose that a reciprocal repartitioning of PKM2-YAP interlinks the cognate metabolic and migrational states of cancer cells; targeting such positive feedback may hold the key to future therapeutic strategies.

cancer biology↗

bioRxiv2025

SP1 amplifies the effects of cellular proliferation via the Wnt/β-catenin pathway in colon development and cancer

Wnt/{beta}-catenin signaling is a highly conserved pathway across multicellular organisms, playing a pivotal role in various cellular processes essential for development and tissue homeostasis. Aberrant activation of this pathway is a major driver of colorectal cancer (CRC). The transcription factor SP1 has been identified as a key component of the Wnt/{beta}-catenin signaling pathway through its physical interaction with {beta}-catenin in colorectal cancer. However, the distinct roles played by this complex during development versus in disease contexts remain poorly understood. In this study, we conducted molecular and functional analyses by individually depleting SP1 and {beta}-catenin. Interestingly, genes co-regulated by the SP1:{beta}-catenin complex were significantly enriched for functions related to the cell cycle and DNA replication--key processes required for sustaining proliferation, whose disruption can promote tumorigenesis. These findings were further supported by data from mouse tumor models and clinical datasets. Additionally, cross-species analysis using zebrafish larvae revealed a synergistic interaction between SP1 and {beta}-catenin, leading to excessive cell proliferation and developmental defects. Collectively, our results suggest a conserved role for the SP1:{beta}-catenin complex in regulating cell proliferation during development and maintaining cellular homeostasis, processes critical for preventing cancer progression.

cancer biology↗

bioRxiv2025

Desiccation-induced DNA damage facilitates drug resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen that depends on its ability to spread from host-to-host to survive as a species. Yet, knowledge of transmission-specific traits remains lacking. Here, we report the discovery of a specific adaptive response to desiccation, a stress intrinsically linked to the generation of the aerosol droplets within which Mtb transmits. We show that desiccation inflicts oxidative damage and activates Mtbs DNA repair responses but that this repair is imperfect and results in mutations. We further show that activation of these DNA repair responses is accompanied by increased expression of the transcription-coupled repair factor, mfd, but that this expression serves to buffer the fitness cost of specific resistance-conferring mutations in rpoB, the target of the frontline drug rifampin, rather than to facilitate transcription-coupled DNA repair. Silencing mfd during aerosolization impairs survival of strains harboring the rifampin resistance allele S450L. This function is further supported by whole genome sequence data from over 50,000 clinically circulating strains. These studies indicate that Mtb has evolved transmission-specific stress responses that have enabled it to leverage desiccation-induced DNA damage as a potential source of genetic diversification and drug resistance.

microbiology↗

bioRxiv2025

A Plasmodium lipase is critical for the disruption of the parasitophorous vacuole membrane and egress of hepatic merozoites.

Malaria parasites develop within a specialized vacuole delimited by the parasitophorous vacuolar membrane (PVM). The exit of Plasmodium from host cells is a well-regulated process involving several molecules that disrupt the PVM to continue the life cycle. While several protein classes play a role in Plasmodium PVM rupture and merozoite egress, the first class of enzymes one would consider to act on membranes is lipases. However, the role of a lipase in PVM disruption remains unknown. Here, we characterized the role of UIS28 (upregulated in infective sporozoite gene 28) in Plasmodium berghei. Bioinformatic studies indicated that UIS28 is a class 3 lipase containing a fungal lipase-like domain. We found that UIS28 localizes to the PVM in infected hepatocytes. To understand the role of UIS28 in PVM disruption, we validated its lipase activity and found that it breaks down lipids into glycerol. Parasites lacking UIS28 develop normally in the blood and mosquito stages and mature fully into hepatic merozoites but show a defect in egress from host hepatocytes. We quantified the PVM rupture and reported that mutant parasites exhibit delayed egress due to impaired PVM disruption. Together, our results demonstrate that UIS28 is a lipase involved in the disruption of the PVM and the successful egress of hepatic merozoites. Author SummaryAn essential step in the life cycle of malaria parasites is their egress from hepatocytes, allowing them to transition from the liver stage to the blood stage. Liver stage parasites reside in a PV, which is surrounded by a PVM. Parasites must breakdown the PVM before they can infect other cells. This complex, multistep process is orchestrated by the parasite and involves several molecules to disrupt the PVM. However, little is known about these molecules. Here, we validated that UIS28 is indeed a lipase. We show that UIS28 is expressed in sporozoites and in the liver stage, and that it localizes to the PVM of parasites within hepatocytes. Disruption of UIS28 gene does not affect blood and mosquito stage development. However, mutant parasites show a defect in the disruption of the PVM and egress of hepatic merozoites. For the first time, we demonstrate the role of a lipase in PVM rupture during the release of liver-stage merozoites.

microbiology↗

bioRxiv2025

Molecular fingerprints of a convergent mechanism orchestrating diverse ligand recognition and species-specific pharmacology at the complement anaphylatoxin receptors

Complement anaphylatoxin receptors (C3aR and C5aR1) are prototypical G protein-coupled receptors (GPCRs) playing crucial physiological roles in innate immunity by combating pathogenic infections and orchestrating inflammatory responses. They continue to be important therapeutic targets for multiple disorders including autoimmune diseases, acute and chronic inflammation, and allergy-related conditions. Recent structural coverage has provided important insights into their activation and signaling, however, confounding observations in the literature related to ligand efficacy and functional responses, especially in different model systems, present a major challenge for drug discovery efforts. Here, we systematically and comprehensively profile a broad set of natural and synthetic ligands at C3aR and C5aR1 and discover a previously unanticipated level of functional specialization in terms of species-specific pharmacology and receptor activation. Taking a lead from this, we determine seventeen cryo-EM structures of different ligand-receptor-G-protein complexes and uncover distinct orientation of agonists between the human and mouse receptors despite an overlapping positioning in the orthosteric binding pocket. Combined with extensive mutagenesis and functional assays, these structural snapshots allow us to decode and validate a convergent molecular mechanism involving a "Five-Point-Switch" in these receptors that orchestrates the recognition and efficacy of diverse agonists. We also identify species-specific differences at the level of phosphorylation patterns encoded in the carboxyl-terminus of these receptors and directly visualize their impact on {beta}arr binding and activation using cryo-EM structures. Interestingly, we observe that {beta}arrs engage with the mouse C5aR1 using a variation of previously discovered P-X-P-P phosphorylation motif via a "Sliding-Mechanism" and also exhibit distinct oligomeric state for the human vs. mouse receptors. Taken together, this study elucidates functional specialization at the complement anaphylatoxin receptors and underlying molecular mechanisms, offering a previously lacking framework with direct and immediate implications for the development of novel therapeutics.

biochemistry↗

bioRxiv2025

In vivo guanine quadruplex structure dynamics and role in genome maintenance in Deinococcus radiodurans

Guanine quadruplex (G4) structures are secondary structures formed in nucleic acids that are rich in guanine bases. G4 structures regulate various cellular processes both in eukaryotes and prokaryotes. Deinococcus radiodurans, an extremophile show sensitivity to ionizing radiation in the presence of G4 binding ligands during the post-irradiation recovery period (PIR). Putative G4 motifs positioned at different locations on the genes fold into different topologies in vitro in the presence of monovalent cations while divalent cation Mg+2 supported the stable G4 formation but Mn+2 destabilizes the G4 structures. Thioflavin T and anti-DNA quadruplex antibodies detected the in vivo formation and dynamics of G4 structure in response to various DNA damaging agents, more in the presence of gamma radiation treatment. G4 binding drugs during PIR arrested the G4 structure dynamics and delayed the DNA repair process. Further, the absence of G4 helicase, RecQ resulted in the accumulation of more G4 structures and genome instability. All these results indicate that the guanine quadruplex structure formation increases in response to cellular stress and G4s are crucial for stable genome maintenance in Deinococcus radiodurans.

microbiology↗

bioRxiv2025

LncRNA Dio3os regulates neighboring gene Dio3 and impacts osteogenesis in trans

Long non-coding RNAs (lncRNAs) frequently act at imprinted loci, yet how a single lncRNA can simultaneously activate its neighbor and repress distant genes through a shared chromatin platform remains poorly defined. We show that imprinted Dio3os performs this bifunctional role by scaffolding the nucleosome remodeling and histone deacetylase (NuRD-HDAC1) complex. ATAC-seq and ChIP-seq across osteoblast differentiation reveal that the Dio3-Dio3os bidirectional promoter is dynamically remodeled and co-occupied by HDAC1, HIF1, and active histone marks. CRISPR-mediated exon deletion, CRISPRi, and polyadenylation termination demonstrate that Dio3os activates Dio3 in cis while repressing osteogenic genes in trans. RIP-MS and reverse pulldown identify direct Dio3os-NuRD interaction; ChIP shows the same machinery produces opposing H3K27ac outcomes at Dio3os versus osteogenic promoters. In vivo, osteoblast-specific CRISPRi increases trabecular and cortical bone mass, whereas CRISPRa reduces it in both sexes. Dio3os couples chromatin remodeling to thyroid-hormone metabolism through a context-dependent NuRD scaffold. One sentence summaryDio3os scaffolds NuRD-HDAC1 to activate Dio3 in cis and repress thyroid-hormone-responsive osteogenic genes in trans.

molecular biology↗

bioRxiv2025

Development of a thermostable and broadly neutralizing pan-sarbecovirus vaccine candidate

Zoonotic spillover of sarbecoviruses to humans resulted in theSARS-CoV-1 outbreak in 2003 and the current COVID-19 pandemic caused by SARS-CoV-2. In both cases, the viral spike protein (S) is the principal target of neutralising antibodies that prevent infection. Within spike, the immunodominant receptor-binding domain (RBD) is the primary target of neutralising antibodies in COVID-19 convalescent sera and vaccine recipients. We have constructed stabilized RBD derivatives of different sarbecoviruses: SARS-CoV-1 (Clade 1a), WIV-1 (Clade 1a), RaTG13 (Clade 1b), RmYN02 (Clade 2) and BtKY72 (Clade 3). Stabilization enhanced yield by an 3-23-fold. The RBD derivatives were conformationally intact as assayed by binding to multiple broadly neutralizing antibodies. The stabilized RBDs show significant enhancement in apparent Tm, exhibit resistance to a 2-hour incubation at temperatures up to 60 in PBS in contrast to corresponding WT RBDs, and show prolonged stability of over 15 days at 37 after lyophilization. In mice immunizations, both stabilization and trimerization significantly enhanced elicited neutralization titers by [~]100 fold. The stabilized RBD cocktail elicited high neutralizing titers against both homologous and heterologous pseudoviruses. The immunogenicity of the vaccine formulation was assessed in both naive and SARS-CoV-2 pre-immunized mice, revealing an absence of immune imprinting, thus indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics. Author summaryThe COVID-19 pandemic was caused by the sarbecovirus SARS-CoV-2. Phylogenetically, sarbecoviruses are divided into four clades: Clade 1a, Clade 1b, Clade 2 and Clade 3 and within these clades, are many other sarbecovirus strains with pandemic or epidemic potential. It is therefore important to develop a broadly protective, pan-sarbecovirus vaccine formulation that can be cheaply and rapidly produced. While mRNA vaccine formulations are efficacious, they have stringent low temperature storage requirements and there is limited manufacturing expertise in low and middle income countries for this modality. Neutralizing antibodies are important for protection and in the case of SARS-CoV-2 are primarily directed against the Receptor Binding Domain (RBD) of the surface spike protein. In this study, we have designed and developed an adjuvanted, protein subunit, pan sarbecovirus vaccine formulation using stabilized RBD derivatives from diverse sarbecoviruses as immunogens. We demonstrate that the stabilized RBD derivatives have considerably enhanced yield and thermal stability relative to corresponding WT proteins and that the formulation remains stable for up to two weeks at 37{degrees}C. The formulation was found to be highly immunogenic in both naive and pre-immunized mice, eliciting neutralizing titers well above the known protective threshold, indicating its suitability for use in future sarbecovirus-origin epidemics or pandemics.

immunology↗

bioRxiv2025

Targeting CHEK2-YBX1&YBX3 regulatory hub to potentiate immune checkpoint blockade response in gliomas

Although GBMs immunosuppressive environment is well known, the tumors resistance to CD8+ T cell killing is not fully understood. Our previous study identified Checkpoint Kinase 2 (Chek2) as the key driver of CD8+ T cell resistance in mouse glioma through an in vivo CRISPR screen and demonstrated that Chk2 inhibition, combined with PD-1/PD-L1 blockade, significantly enhanced CD8+ T cell-mediated tumor killing and improved survival in preclinical model. Here, we aimed to elucidate the immunosuppressive function of Chek2. Immunoprecipitation (IP) followed by mass spectrometry (MS) and phosphoproteomics identified an association between Chek2 with the DNA/RNA-binding proteins YBX1 and YBX3 that are implicated in transcriptional repression of pro-inflammatory genes. Single-gene knock-out and overexpression studies of CHEK2, YBX1, and YBX3 in multiple glioma cell lines revealed that these proteins positively regulate each others expression. RNA sequencing coupled with chromatin immunoprecipitation-sequencing (ChIP-seq) analysis demonstrated common inflammatory genes repressed by CHK2-YBX1&YBX3 hub. Targeting one of the hub proteins, YBX1, with the YBX1 inhibitor SU056 led to degradation of CHK2-YBX1&YBX3 hub. Targeting of this hub by SU056 led to enhanced antigen presentation and antigen specific CD8+ T cell proliferation. Further, combination of SU056 with ICB significantly improved survival in multiple glioma models. Collectively, these findings reveal an immunosuppressive mechanism mediated by the CHK2-YBX1&YBX3 hub proteins. Therefore, CHK2-YBX1&YBX3 hub targeting in combination with immune checkpoint blockade therapies in gliomas is warranted.

cancer biology↗

bioRxiv2025

Plasmodium actin-like proteins are essential for DNA segregation during male gametogenesis and malaria transmission

Protozoan parasites of the genus Plasmodium cause malaria and involve infection of multiple hosts and cell types during the life cycle. Producing sexually fit gametocytes is essential for transmitting the Plasmodium parasite into an anopheline mosquito vector. After the uptake of malaria parasites, male gametocytes undergo three rounds of DNA replication to produce eight nucleated flagellar gametes. Here, we report that the actin-like proteins Alp5a and Alp5b are involved in DNA segregation during male gametogenesis. The Plasmodium-specific paralogous genes Alp5a and Alp5b superimposed on human Arp2 and Arp3, localize to the nucleus, and interact with each other. Alp5a and Alp5b are dispensable for the development of P. berghei blood-stages individually but indispensable simultaneously. In agreement with genetic studies, the inhibitory activity of the Arp2/3 complex inhibitor in Plasmodium supports an essential role for this complex during the blood stage. Deletion of Alp5a or Alp5b did not affect actin nucleation, parasite growth, or gametocytemia in the blood. The knockout parasites successfully invaded the midgut and developed into oocysts, but their size was significantly reduced, and they failed to survive. Genetic crosses revealed defects in male gamete integrity. We found that the reduced oocyst development was due to impaired DNA segregation during male gametogenesis. This study provides molecular insight into the fundamental requirements of the Alps in Plasmodium, which are essential for malaria transmission.

microbiology↗

bioRxiv2025

Candidate transmission survival genome of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), a leading cause of death from infection, completes its life cycle entirely in humans except for transmission through the air. To begin to understand how Mtb survives aerosolization, we mimicked liquid and atmospheric conditions experienced by Mtb before and after exhalation using a model aerosol fluid (MAF) based on the water-soluble, lipidic and cellular constituents of necrotic tuberculosis lesions. MAF induced drug tolerance in Mtb, remodeled its transcriptome and protected Mtb from dying in microdroplets desiccating in air. Yet survival was not passive: Mtb appeared to rely on hundreds of genes to survive conditions associated with transmission. Essential genes subserving proteostasis offered most protection. A large number of conventionally nonessential genes appeared to contribute as well, including genes encoding proteins that resemble anti-desiccants. The candidate transmission survival genome of Mtb may offer opportunities to reduce transmission of tuberculosis. Significance StatementMycobacterium tuberculosis (Mtb) travels from the lungs of one person through the air to the lungs of another and survives multiple stresses en route, including changes in temperature and in concentrations of oxygen, carbon dioxide, hydrogen ions, salts and organic solutes. Here we present a genetically tractable model of transmission to begin the identification of the transmission survival genome of Mtb. We devised a fluid that mimics TB lesions, found that it protects Mtb from transmission-related stresses, associated this with the structure of the droplets as they dry and their ability to retain water, and used it to query the potential contribution of each of Mtbs genes to Mtbs survival in models of three sequential stages of transmission.

microbiology↗

bioRxiv2024

Horizontal Gene Transfer Inference: Gene presence-absence outperforms gene trees

Horizontal gene transfer (HGT) is a fundamental driver of prokaryotic evolution, facilitating the acquisition of novel traits and adaptation to new environments. Despite its importance, methods for inferring HGT are rarely systematically compared, leaving a gap in our understanding of their relative strengths and limitations. Validating HGT inference methods is challenging due to the absence of a genomic fossil record that could confirm historical transfer events. Without an empirical gold standard, new inference methods are typically validated using simulated data; however, these simulations may not accurately capture biological complexity and often embed the same assumptions used in the inference methods themselves. Here, we leverage the tendency of HGT events to involve multiple neighboring genes to assess the accuracy of diverse HGT inference methods. We show that methods analyzing gene family presence/absence patterns across species trees consistently outperform approaches based on gene tree-species tree reconciliation. Our findings challenge the prevailing assumption that explicit phylogenetic reconciliation methods are superior to simpler implicit methods. By providing a comprehensive bench-mark, we offer practical recommendations for selecting appropriate methods and indicate avenues for future methodological advancements.

bioinformatics↗

bioRxiv2024

Horizontal gene transfer rates decrease during transitions to new ecosystems

Bacterial colonization of unfamiliar environments constitutes a drastic evolutionary transition, likely altering the strength and direction of selection. This process is often assumed to increase rates of horizontal gene transfer (HGT), a major driver of bacterial adaptation. Larger genomes are thought to facilitate such adaptations by providing broader functional repertoires and more integration sites for foreign DNA. Here, we systematically test these ideas across a broad bacterial phylogeny linked to environmental transitions inferred from metagenomic data. Contrary to expectation, we find that bacteria entering new environments typically have smaller genomes and experience lower rates of HGT. The reduction in HGT is fully explained by genome size, with no residual effect of environmental transitions once size is controlled for. These findings suggest that successful bacterial colonizers rely less on genomic plasticity through HGT than previously assumed, highlighting gaps in our understanding of microbial evolutionary dynamics.

bioinformatics↗

bioRxiv2024

GEMS: A Generalizable GNN Framework For Protein-Ligand Binding Affinity Prediction Through Robust Data Filtering and Language Model Integration

The field of computational drug design requires accurate scoring functions to predict binding affinities for protein-ligand interactions. However, train-test data leakage between the PDBbind database and the CASF benchmark datasets has significantly inflated the performance metrics of currently available deep-learning-based binding affinity prediction models, leading to overestimation of their generalization capabilities. We address this issue by proposing PDBbind CleanSplit, a training dataset curated by a novel structure-based filtering algorithm that eliminates train-test data leakage as well as redundancies within the training set. Retraining current top-performing models on CleanSplit caused their benchmark performance to drop significantly, indicating that the performance of existing models is largely driven by data leakage. In contrast, our graph neural network model for efficient molecular scoring (GEMS) maintains high benchmark performance when trained on CleanSplit. Leveraging a sparse graph modeling of protein-ligand interactions and transfer learning from language models, GEMS is able to generalize to strictly independent test datasets.

bioinformatics↗

bioRxiv2024

A TORC1-PHR1 signaling axis regulates phosphorus starvation and immunity signaling network in Arabidopsis

The Target of Rapamycin Complex 1 (TORC1) is a crucial eukaryotic kinase that modulates growth in response to nutrient availability. Phosphorus (P) is an essential macronutrient, and its deficiency induces extensive reprogramming of growth and defense strategies in plants. This process involves Phosphate Starvation Response 1 (PHR1), a master regulator of the Phosphate Starvation Response (PSR). In this study, we identify a novel, non-canonical role for TORC1 in regulating P starvation responses in Arabidopsis. We demonstrate that P limitation activates TORC1, leading to the stabilization of PHR1. Inhibition of TORC1 increased sensitivity to P starvation, accompanied by disruption of starvation-induced transcriptional reprogramming. Additionally, our results reveal that the TORC1-PHR1 signaling axis plays a crucial role in reprogramming the expression of genes involved in the plant immune signaling network. This regulation is critical for the symbiotic association with the endophytic fungus Piriformospora indica under P starvation. These findings underscore the significant role of the TORC1-PHR1 module in orchestrating the PSR and highlight the evolutionary adaptation of TORC1 signaling pathways in plants.

plant biology↗

bioRxiv2024

p-ClustVal: A Novel p-adic Approach for Enhanced Clustering of High-Dimensional scRNASeq Data

This paper introduces p-ClustVal, a novel data transformation technique inspired by p-adic number theory that significantly enhances cluster discernibility in genomics data, specifically Single Cell RNA Sequencing (scRNASeq). By leveraging p-adic-valuation, p-ClustVal integrates with and augments widely used clustering algorithms and dimension reduction techniques, amplifying their effectiveness in discovering meaningful structure from data. The transformation uses a data-centric heuristic to determine optimal parameters, without relying on ground truth labels, making it more user-friendly. p-ClustVal reduces overlap between clusters by employing alternate metric spaces inspired by p-adic-valuation, a significant shift from conventional methods. Our comprehensive evaluation spanning 30 experiments and over 1400 observations, shows that p-ClustVal improves performance in 91% of cases, and boosts the performance of classical and state of the art (SOTA) methods. This work contributes to data analytics and genomics by introducing a unique data transformation approach, enhancing downstream clustering algorithms, and providing empirical evidence of p-ClustVals efficacy. The study concludes with insights into the limitations of p-ClustVal and future research directions.

bioinformatics↗

bioRxiv2024

An endogenous peptide PEP2 modulates Iron-deficiency signalling and root growth in Arabidopsis.

Iron (Fe) is an essential element for most of the living organisms including plants and humans, where plants serve as the primary source of our dietary iron intake. The availability of Fe determines plant fitness and yield. Thus, understanding of iron uptake, its acquisition and utilization is of critical importance to reap nutritional benefits from plant breeding. Despite significant progress in uncovering how iron homeostasis is regulated by transcription factors and phytohormones, molecular pathways that mediate Fe deficiency through the action of signalling peptides remain elusive. In this work, we reported the role of PROPEP2 (Plant Elicitor Peptide 2) and PEPR (Perception of Arabidopsis danger signal peptide receptor) in regulating plant growth and development under Fe-deprived conditions. We revealed that a Damage Associated Molecular Pattern (DAMP) such as PROPEP2 is significantly induced under Fe deficiency. We also show that PEP2 modulates the expression of Iron Regulated Transporter 1 (IRT1) and Ferric-Reduction Oxidase (FRO2) under Fe deficiency. Furthermore, we showed that PEPR2 perceives PEP2 to positively regulate reactive oxygen species (ROS) content and negatively regulate the primary root growth, iron content and rhizosphere acidification. Our findings reveal the complex interplay between Fe and DAMP signalling pathways in plants.

plant biology↗

bioRxiv2024

A human induced pluripotent stem (hiPS) cell model for the holistic study of epithelial to mesenchymal transitions (EMTs)

The epithelial to mesenchymal transition (EMT) is a widely studied but poorly defined state change due to the variety of ways in which it has been characterized in cells. There is a need for reproducible cell model systems that enable the integration and comparison of different types of measured observations of cells across many distinct cellular contexts. We present human induced pluripotent stem (hiPS) cells as such a model system by demonstrating its utility through a comparative analysis of hiPS cell-EMT in 2D and 3D cell culture geometries. We developed live-imaging-based assays to directly compare examples of changes in cell function (via migration timing), molecular components (via expression of marker proteins), organization (via reorganization of cell junctions), and environment (via dynamics of basement membrane) in the same experimental system. The EMT-related changes we measured occurred earlier in 2D colonies than in 3D lumenoids, likely due to differences in the basement membrane environments associated with 2D vs. 3D initial hiPS cell culture geometries. We have made the 449 60-hour-long 3D time-lapse movies and the associated tools used for analysis and visualization open-source and easily accessible as a resource for future work in this field.

cell biology↗