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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2025

A phase oscillator model of cell cycles reveals nuclear density control in a branched fungal network

Multinucleate cells are widespread in biology, from human skeletal muscle and placenta to many filamentous fungi, yet their basic cell biology remains poorly understood. Maintaining an appropriate nuclear-to-cytoplasmic ratio is essential across cell types for physiological function, and mechanisms of size control have been extensively studied in mononucleate cells. Much less is known about how comparable control is achieved in cells where many nuclei share a common cytoplasm. The filamentous fungus Ashbya gossypii forms a branching mycelial network in which individual nuclei divide asynchronously, while the number of nuclei per cell volume (the nuclear density) is tightly controlled. How global regulation of nuclear density coexists with local cell cycle asynchrony remains unclear. We address this by combining a mathematical model of nuclear division in a growing and branching cell with live-cell microscopy. We model nuclei as a dividing population of phase oscillators within a branching cell network and parameterize the model with measurements from Ashbya cells. The model demonstrates that asynchrony is required to prevent large density fluctuations that would result from synchronous division, and that introducing a nuclear-density checkpoint to the cell cycles leads to synchrony if it is the only mechanism of density control. We find that coupling branch formation to nuclear density both stabilizes nuclear density and prevents the emergence of synchronous cycles. Our results indicate that asynchronous nuclear cycles together with density-responsive branching maintain a constant nuclear density, revealing a strategy for regulating the nuclear-to-cytoplasmic ratio in large multinucleate cells. SignificanceMultinucleate cells appear in diverse biological contexts, from human tissues to filamentous fungi, yet many fundamental aspects of their cell biology are still unclear. Regulating the nuclear-to-cytoplasmic ratio is important across cell types, and little is known about how this is achieved in large, multinucleate fungal cells. Using mathematical modeling and live-cell imaging, we identify how the density of nuclei is controlled in a growing and branching fungal mycelial network. We find that asynchronous nuclear cycles together with density-responsive branching can stabilize the nuclear-to-cytoplasmic ratio within a growing fungal network. This illustrates how large multinucleate cells can control nuclear density even as their morphology becomes increasingly complex.

cell biology↗

bioRxiv2025

LazyNet: Interpretable ODE Modeling of Sparse CRISPR Single-Cell Screens Reveals New Biological Insights

We present LazyNet, a compact one-step neural-ODE model for single-cell CRISPR A/I that operates directly on two-snapshot ("pre [->] post") measurements and yields parameters with clear mechanistic meaning. The core log-linear-exp residual block exactly represents multiplicative effects, so synergistic multi-locus responses appear as explicit components rather than opaque composites. On a 53k-cell x 18k-gene neuronal Perturb-seq matrix, a three-replica LazyNet ensemble trained under a matched 1-hour budget achieved strong threshold-free ranking and competitive error (genome-wide r{approx}0.67) while running on CPUs, compared with transformer and state-space baselines trained on a single V100 with the same time cap. A T-cell screen included only for generalization showed the same ranking advantage under the identical evaluation pipeline. Beyond prediction, LazyNet exposes directed, local elasticities; averaging Jacobians across replicas produces a consensus interaction matrix from which compact subgraphs are extracted and evaluated at the module level. The resulting networks show coherent enrichment against authoritative resources (e.g., large-scale co-expression and curated functional associations) and concordance with orthogonal GPX4-knockout proteomes, recovering known ferroptosis regulators and nominating testable links in a lysosomal-mitochondrial-immune module.

bioinformatics↗

bioRxiv2025

Hedyotis Diffusae Herba Mitigates Rotenone-Induced Neurotoxicity via Akt Pathway in SH-SY5Y Cells

Hedyotis Diffusae Herba (HDH), known in traditional Chinese medicine for its heat-clearing and detoxifying properties, has been widely used to manage inflammation-related conditions. However, its neuroprotective potential and mechanism in the context of Parkinsons disease (PD) remain unclear.Network pharmacology and molecular docking were applied to predict the interactions between HDH components and PD-associated targets. Molecular dynamics simulations assessed the binding stability of major compounds to core targets. Rotenone-induced SH-SY5Y cell injury served as an in vitro PD model to evaluate HDHs biological effects. Cell viability, reactive oxygen species (ROS), IL-6 secretion, and Akt phosphorylation were assessed by CCK-8 assay, ROS detection kit, ELISA, and western blot, respectively. MK2206, an Akt inhibitor, was used to validate pathway involvement.Seven bioactive compounds were identified in HDH, among which stigmasterol and quercetin exhibited strong binding affinities with AKT1, IL-6, and JUN. Simulations confirmed stable interactions. HDH significantly increased cell viability and decreased intracellular ROS and IL-6 levels in rotenone-treated SH-SY5Y cells. Akt phosphorylation was restored by HDH, and the effect was blocked by MK2206, indicating Akt pathway participation.HDH exerts neuroprotective effects against rotenone-induced toxicity via antioxidant and anti-inflammatory activities and Akt pathway activation. These findings provide mechanistic insight and pharmacological support for HDH as a potential candidate in complementary therapy for PD.

cell biology↗

bioRxiv2025

Chromatin is a long-range force generator that regulates plasma membrane tension and cell integrity independently of gene expression

Primarily studied for its role in gene expression, chromatin organization is emerging as an important regulator of nuclear mechanics. Although the nucleus is in mechanical equilibrium with the cell, we do not know whether and how chromatin reorganization actively regulates the mechanical properties and downstream behaviors of cells. Here, we tested the hypothesis that as a dynamic crosslinked polymer, chromatin directly impacts cell mechanics independently of transcription by studying NETosis: a transcription-independent process where chromatin decompacts and the plasma membrane (PM) ruptures. Using high resolution microscopy and ATAC-see, we found that chromatin accessibility progressively increases during NETosis suggesting that chromatin binding proteins (CBPs) dissociate from chromatin during NETosis. To determine the identity and dynamics of these dissociated CBPs, we used fluorescent recovery after photobleaching to measure the mobility and localization of the linker histone H1, the nucleosomal histone H3 and the heterochromatin binding protein HP1. We found that the mobile fraction of nuclear H1 increases during NETosis while fractions of HP1 and H3 diffuse outside of the nucleus suggesting that they become cytosolic osmolytes and potentially alter the mechanical state of cells. Consistently, we found that plasma membrane tension and cell volume increase as chromatin decompacts during NETosis. In non-NETing U2OS cells, we found that inducing chromatin decompaction increases plasma membrane tension, independently of the cytoskeleton, indicating a causal relationship between chromatin organization, cell volume and plasma membrane tension. Our findings reveal a novel non- genetic role of chromatin in cellular biophysics: regulating cell volume, PM tension, and thus, overall cell mechanics. Considering the critical role of cell mechanics in biological processes such as cell migration, proliferation and pathogen killing, our work broadens our understanding of how chromatin regulates cell physiology and pathology. Significance StatementChromatin organizes our DNA inside the nucleus and is important for gene expression. However, chromatin is also a polymer which can passively regulate the rigidity of the nucleus, but whether and how chromatin can actively regulate the mechanical properties of the whole cell remains unknown. Here, we leverage the immune process of NETosis to show that the organization of chromatin inside the nucleus actively regulates the volume and tension of cells. Our work establishes chromatin as a long-range force generator in cells, broadening our understanding of the roles of this crucial polymer network in cells and opening the door to new strategies for controlling the mechanical properties of cells as needed by their physiology.

cell biology↗

bioRxiv2015

A cell-free framework for biological systems engineering

SUMMARYWhile complex dynamic biological networks control gene expression and metabolism in all living organisms, engineering comparable synthetic networks remains challenging1,2. Conducting extensive, quantitative and rapid characterization during the design and implementation process of synthetic networks is currently severely limited due to cumbersome molecular cloning and the difficulties associated with measuring parts, components and systems in cellular hosts. Engineering gene networks in a cell-free environment promises to be an efficient and effective approach to rapidly develop novel biological systems and understand their operating regimes3-5. However, it remains questionable whether complex synthetic networks behave similarly in cells and a cell-free environment, which is critical for in vitro approaches to be of significance to biological engineering. Here we show that synthetic dynamic networks can be readily implemented, characterized, and engineered in a cell-free framework and consequently transferred to cellular hosts. We implemented and characterized the \"repressilator\"6, a three-node negative feedback oscillator in vitro. We then used our cell-free framework to engineer novel three-node, four-node, and five-node negative feedback architectures going from the characterization of circuit components to the rapid analysis of complete networks. We validated our cell-free approach by transferring these novel three-node and five-node oscillators to Escherichia coli, resulting in robust and synchronized oscillations reflecting the in vitro observation. We demonstrate that comprehensive circuit engineering can be performed in a cell-free system and that the in vitro results have direct applicability in vivo. Cell-free synthetic biology thus has the potential to drastically speed up design-build-test cycles in biological engineering and enable the quantitative characterization of synthetic and natural networks.

Synthetic Biology↗

bioRxiv2020

Decision-making in a synthetic cell: the limits of biological computation

We measured the dynamics of decision-making by a minimal bistable gene network integrated in a synthetic cell model, free of external perturbations. Reducing the number of gene copies from 105 to about 10 per cell revealed a transition from deterministic and slow computation to a fuzzy and rapid regime dominated by singleprotein fluctuations. Fuzzy computation appeared at DNA and protein concentrations 100-fold lower than necessary in equilibrium, suggesting rate enhancement by co-expressional localization. Whereas the high-copy regime was characterized by a sharp transition, hysteresis and robust memory, the low-copy limit showed incipient strong fluctuations, switching between states, and a signature of cellular individuality across the decision-making point. Our work establishes synthetic cells operating rapidly at the single molecule level to integrate gene regulatory networks with metabolic pathways for sustained survival with low energetic cost.One Sentence Summary Decision-making in a synthetic cell can be slow and precise or rapid and probabilistic by reducing the number of computing molecules by five decades down to single-molecule fluctuations.Competing Interest StatementThe Noireaux laboratory receives research funds from Arbor Biosciences, a distributor of the myTXTL cell-free protein synthesis kit.View Full Text

synthetic biology↗

bioRxiv2022

Multi-modal quantification of pathway activity with MAYA

Signaling pathways can be activated through various cascades of genes depending on cell identity and biological context. Single-cell atlases now provide the opportunity to inspect such complexity in health and disease. Yet, existing reference tools for pathway scoring resume activity of each pathway to one unique common metric across cell types. Here, we present MAYA a computational method that enables the automatic detection and scoring of the diverse modes of activation of biological pathways across cell populations. MAYA improves the granularity of pathway analysis by detecting subgroups of genes within reference pathways, each characteristic of a cell population and how it activates a pathway. Using multiple single-cell datasets, we demonstrate the biological relevance of identified modes of activation, the robustness of MAYA to noisy pathway lists and batch effect. MAYA can also predict cell types starting from lists of reference markers in a cluster-free manner. Finally, we show that MAYA reveals common modes of pathway activation in tumor cells across patients, opening the perspective to discover shared therapeutic vulnerabilities.

bioinformatics↗

bioRxiv2024

High resolution live cell imaging to define ultrastructural and dynamic features of the halotolerant yeast Debaryomyces hansenii

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and different membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

cell biology↗

bioRxiv2020

Tripartite separation of glomerular cell-types and proteomes from reporter-free mice

PurposeThe kidney glomerulus comprises a syncytium of podocytes, mesangial and endothelial cells, which jointly determine glomerular filtration barrier function, and thereby kidney and cardiovascular health. The understanding of this intricate functional unit and its intracellular communication beyond the transcriptome requires bulk isolation of these cell-types from glomeruli for subsequent biochemical investigations. Therefore, we developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). MethodsGlomerular cells were separated via a novel FACS-sort depending on a cell-specific antibody labeling in wildtype mice or based on a combination of transgenic fluorescent protein expression and antibody labeling in mT/mG mice. The purity of isolated cell-types was validated by qPCR and immunoblot. The proteome of podocytes, mesangial and endothelial cells was determined and compared between species, ages and gender of wildtype and mT/mG mice. The method was also applied to the podocyte-targeting immunologic injury model of THSD7A-associated membranous glomerulonephritis. ResultsTimMEP enabled protein-biochemical analyses of podocytes, mesangial and endothelial cells derived from a single reporter free mouse. Proteomic analyses allowed the first characterization of podocyte, endothelial and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were determined, and protein-based interaction and intraglomerular cell communication networks were elucidated. Interestingly, analyses revealed significant cell-type specific proteome differences between mouse strains, artefacts induced by reporters, and alterations depending on gender and age. Within the glomerulus, timMEP resolved a fine-tuned initial stress response exclusively in podocytes after exposure to anti-THSD7A antibodies, which was not detectable using conventional analyses in whole glomeruli. ConclusionGlobally applicable timMEP abolishes the need for costly, time- and animal-consuming breeding of mice to glomerular cell-type reporters. TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein biochemical level in health and disease, while avoiding reporter-based artefacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology. Key messagesO_LIA tripartite isolation method for mesangial, endothelial and podocyte cell-types from reporter-free mice. C_LIO_LIGeneration of bulk cell-type samples and primary co-cultures for biochemical and protein-based analyses. C_LIO_LIStrain and transgene-dependent expression of proteins among glomerular cell-types, including protein profiles, intra-glomerular communication machineries, and reporter-dependent artefacts. C_LIO_LIDisease specific time-resolved resolution of glomerular cell-types response to injury. C_LI

cell biology↗

bioRxiv2020

Using Expansion Microscopy to visualize and characterize the morphology of mitochondrial cristae

Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signalling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to ageing and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED and SIM have been applied for cristae visualisation. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4-4.5, improving the resolution to 60-70 nm on conventional confocal microscopes, which can be further increased to [~] 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labelled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labelling and ExM as a tool for studying internal structure of mitochondria.

cell biology↗

bioRxiv2021

Core cysteine residues in the PAN domain are critical for HGF/c-MET signaling

The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in > 28,000 proteins across 959 genera but its role in protein function is not fully understood. The PAN domain was initially characterized to be present in numerous proteins including hepatocyte growth factor (HGF). Dysregulation of HGF-mediated signaling results in numerous deadly cancers. All biological impacts of HGF in cell proliferation are triggered by binding of HGF to its cell surface receptor, cellular mesenchymal-epidermal transition (c-MET). Here, we show that four PAN domain cysteine residues are essential for HGF/c-MET signaling. Mutating these residues resulted in retardation of perinuclear localization, cellular internalization of HGF and its receptor, c-MET, and c-MET ubiquitination. Our observations indicate that the PAN domain of HGF is required for the c-MET binding and subsequent c-MET autophosphorylation and phosphorylation of its downstream targets, protein kinase B (AKT), extracellular signal-regulated kinase (ERK), and signal transducer and activator of transcription 3 (STAT3). Furthermore, transcriptional activation of HGF/c-MET signaling-related genes including matrix metalloproteinase-9 (MMP9), ETS translocation variant 1, 4, and 5 (ETV1, ETV4, ETV5), and early growth response 1 (EGR1) was impaired and cell proliferation was attenuated. These results suggest that core cysteine residues in the PAN domain are critical for HGF/c-MET interaction, c-MET mediated signal transduction, and cell survival. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway. SignificanceHGF/c-MET signaling induces multifunctional cellular responses. Dysregulation of HGF/c-MET signaling cascade can lead to tumorigenesis by transforming normal cells to tumor cells. This work defines the importance of core cysteine residues in the PAN domain of HGF in downstream activation of HGF/c-MET signaling. To understand the role of cysteines in the PAN domain, PAN mutants of HGF were used to stimulate c-MET signaling in cells and the impact was delineated by determining phosphorylation and transcription of downstream targets. Mutations in core cysteines in the HGF-PAN domain completely blocked downstream phosphorylation and perinuclear accumulation of c-MET. These results suggest an indispensable role for the cysteine-rich PAN domain in HGF/c-MET interaction and could set the stage for future therapies that selectively disrupt the MET signaling cascade with limited off-target effects in tumors overexpressing HGF/c-MET.

cell biology↗

bioRxiv2022

Uncovering context-specific genetic-regulation of gene expression from single-cell RNA-sequencing using latent-factor models

Genetic regulation of gene expression is a complex process, with genetic effects known to vary across cellular contexts such as cell types and environmental conditions. We developed SURGE, a method for unsupervised discovery of context-specific expression quantitative trait loci (eQTLs) from single-cell transcriptomic data. This allows discovery of the contexts or cell types modulating genetic regulation without prior knowledge. Applied to peripheral blood single-cell eQTL data, SURGE contexts capture continuous representations of distinct cell types and groupings of biologically related cell types. We demonstrate the disease-relevance of SURGE context-specific eQTLs using colocalization analysis and stratified LD-score regression.

bioinformatics↗

bioRxiv2025

Proteomic Signatures of Podocyte Injury Are Reflected in Urinary Extracellular Vesicles in Pediatric Nephrotic Syndrome.

Idiopathic nephrotic syndrome (NS) is a common glomerulopathy in children and presents with significant proteinuria. There are no reliable clinical or biochemical markers of disease relapse, or prognosis. Extracellular vesicles (EVs) are small, membrane-bound biological effectors released from stressed cells. We previously showed increases in podocyte-specific urinary EVs from children with disease relapse in NS, with numbers returning to near-zero in remission. Herein, we have expanded this work to evaluate puromycin aminonucleoside (PAN) injury by characterizing proteomic signatures of podocytes and their EVs in vitro. In addition, we performed data-independent proteomic analysis (DIA) to characterize changes in signatures of EVs from pediatric patients with active NS versus remission. Our key findings reveal PAN-injured podocytes increase large EV (LEV) secretion in vitro; moreover, DIA uncovered changes in cellular and LEV proteomes that were also observed in urinary LEVs from patients with active disease. Urinary LEV proteomes from children with active NS were significantly different than those in remission, highlighted by 645 and 240 unique proteins associated with disease or remission, respectively. This foundational work provides the impetus for a larger, prospective biomarker study aimed at identifying EV-specific proteins associated with relapse versus remission.

cell biology↗

bioRxiv2018

Identification of a putative nuclear localization signal in maspin protein shed light into its nuclear import regulation

Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies suggest that subcellular localization plays an essential role on maspin tumor suppression activity. In this study we investigated the molecular mechanisms underlying maspin nucleocytoplasmic shuttling. An in vitro nuclear-import assay using digitonin-permeabilized HeLa cells demonstrated that maspin enters the nucleus by an energy-and carrier-independent mechanism. However, previous studies indicated that maspin subcellular localization is regulated in the cell. Using a nuclear localization signal (NLS) prediction software, we identified a putative NLS in the maspin amino acid sequence. To distinguish between passive and regulated nuclear translocation, maspinNLS or the full-length protein (MaspinFL) were fused to 5GFP, rendering the construct too large to enter the nucleus passively. Unexpectedly, 5GFP-maspinNLS, but not maspinFL-5GFP, entered the nucleus of HeLa cells. Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N, suppressed 5GFP-maspinNLS nuclear localization. In summary, we provide evidence that maspin translocates to the nucleus passively. In addition, we identified a peptide in the maspin protein sequence, which is able to drive a 5GFP construct to the nucleus in an energy-dependent manner.

cell biology↗

bioRxiv2026

A bone fragment-based protocol for molecular analysis of osteocyte-associated transcripts in human bone specimens

Osteocytes play a central role in bone remodeling, mineral metabolism, and skeletal homeostasis, but direct molecular analysis of human osteocytes remains technically challenging because they are embedded within the mineralized bone matrix. Surgically obtained human bone specimens provide valuable material for studying human bone biology; however, surface-associated cells, marrow-derived cells, and adherent soft tissues can confound downstream transcript analysis. Here, we describe a bone fragment-based protocol for preparing surgically obtained human bone specimens for molecular analysis of osteocyte-associated transcripts. The protocol consists of mechanical trimming, mincing into small bone fragments, repeated washing, and five sequential rounds of collagenase digestion to reduce non-osteocytic cellular components associated with the bone surface and marrow spaces. The remaining mineralized bone fragments are then frozen in liquid nitrogen, cryogenically pulverized, and lysed in TRIzol reagent for total RNA extraction. Histological validation using residual maxillary bone specimens showed that sequential collagenase digestion markedly reduced adherent soft tissue and extra-matrix nuclei while preserving osteocyte lacunar occupancy. This protocol provides a practical workflow for bone fragment-based RNA analysis focused on osteocyte-associated transcripts in human bone specimens. Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@1cec618org.highwire.dtl.DTLVardef@2f746forg.highwire.dtl.DTLVardef@1854247org.highwire.dtl.DTLVardef@1c26c1aorg.highwire.dtl.DTLVardef@1473a88_HPS_FORMAT_FIGEXP M_TBL C_TBL

cell biology↗

bioRxiv2018

BioLogic, a parallel approach to cell-based logic gates

AbstractIn vivo logic gates have proven difficult to combine into larger devices. Our cell-based logic system, BioLogic, decomposes a large circuit into a collection of small subcircuits working in parallel, each subcircuit responding to a different combination of inputs. A final global output is then generated by a combination of the responses. Using BioLogic, for the first time a completely functional 3-bit full adder and full subtractor were generated using Escherichia coli cells; as well as a calculator-style display that shows a numeric result, from 0 to 7, when the proper 3 bit binary inputs are introduced into the system. BioLogic demonstrates the use of a parallel approach for the design of cell-based logic gates that facilitates the generation and analysis of complex processes, without the need for complex genetic engineering.

synthetic biology↗

bioRxiv2018

DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, also several affinity reagents against fluorescent proteins have been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfill their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.\n\nSummary statementStructural characterization of two novel DARPins (designed ankyrin repeat proteins) recognizing the monomeric teal fluorescent protein 1 (mTFP1) and their functionalization for protein manipulation strategies in cultured cells and potentially in living organisms.

cell biology↗

bioRxiv2026

TRAILBLAZER: generative multicellular perturbation model of biology

Single-cell foundation models are reshaping biology by learning transferable representations of cellular state from millions of profiles. These models support annotation, denoising, cross-modal mapping and, increasingly, prediction of responses to genetic or pharmacological perturbations. Despite this progress, most approaches treat cells as independent observations and ignore the multicellular context that governs tissue behavior. Models trained on aggregated datasets often fail to generalize to new donors, laboratories or interventions, in part because their latent spaces lack structure for composition and extrapolation. As a result, strong reconstruction performance does not guarantee accurate forecasting of system-level responses. The general problem addressed here is how to construct a scalable model that predicts multicellular, patient-level responses to interventions while preserving single-cell resolution and enabling generalization beyond observed conditions. Here we show that TRAILBLAZER, a multicellular transformer encoder coupled to an explicitly shaped hyperspherical latent space and a count-aware generative decoder, enables accurate zero-shot prediction of perturbation responses and ranking of candidate immunomodulators at patient resolution. In contrast to prior single-cell or pseudo-bulk approaches, TRAILBLAZER models tissues as coordinated systems using latent tokens that summarize and redistribute global context while maintaining near-linear scaling with group size. By organizing latent geometry around shared healthy references and calibrated mechanistic directions, the model renders vector arithmetic biologically meaningful and supports extrapolation to unseen agents. Together, these results establish a practical framework for mechanism-aware simulation of multicellular responses and suggest a path toward predictive foundation models for therapeutic discovery.

systems biology↗