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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2020

The C-terminal actin binding domain of talin forms an asymmetric catch bond with F-actin

Focal adhesions (FAs) are large, integrin-based adhesion complexes that link cells to the extracellular matrix (ECM). Previous work demonstrates that FAs form only when and where they are necessary to transmit force between the cellular cytoskeleton and the ECM, but how this occurs remains poorly understood. Talin is a 270 kDa adapter protein that links integrins to filamentous (F)-actin and recruits additional components during FA assembly in a force-dependent manner. Cell biological and developmental data demonstrate that the third, and C-terminal, F-actin binding site (ABS3) of talin is required for normal FA formation. However, ABS3 binds F-actin only weakly in in vitro, biochemical assays. We used a single-molecule optical trap assay to examine how and whether ABS3 binds F-actin under physiologically relevant, pN mechanical loads. We find that ABS3 forms a directional catch bond with F-actin when force is applied towards the pointed end of the actin filament, with binding lifetimes more than 100-fold longer than when force is applied towards the barbed end. Long-lived bonds to F-actin under load require the ABS3 C-terminal dimerization domain, whose cleavage is known to regulate focal adhesion turnover. Our results support a mechanism in which talin ABS3 preferentially binds and orients actin filaments with barbed ends facing the cell periphery, thus nucleating long-range order in the actin cytoskeleton. We suggest that talin ABS3 may function as a molecular AND gate that allows FA growth only when sufficient integrin density, F-actin polarization, and mechanical tension are simultaneously present.

biophysics↗

bioRxiv2022

A Caenorhabditis elegans nck-1 and filamentous actin-regulating protein pathway mediates a key cellular defense against bacterial pore-forming proteins

Pore-forming proteins (PFPs) comprise the largest single class of bacterial protein virulence factors and are expressed by many human and animal bacterial pathogens. Cells that are attacked by these virulence factors activate epithelial intrinsic cellular defenses (or INCEDs) to prevent the attendant cellular damage, cellular dysfunction, osmotic lysis, and organismal death. Several conserved PFP INCEDs have been identified using the nematode Caenorhabditis elegans and the nematicidal PFP Cry5B, including mitogen-activated protein kinase (MAPK) signaling pathways. Here we demonstrate that the gene nck-1, which has homologs from Drosophila to humans and links cell signaling with localized F-actin polymerization, is required for INCED against small-pore PFPs in C. elegans. Reduction/loss of nck-1 function results in C. elegans hypersensitivity to PFP attack, a hallmark of a gene required for INCEDs against PFPs. This requirement for nck-1-mediated INCED functions cell-autonomously in the intestine and is specific to PFPs but not other tested stresses. Genetic interaction experiments indicate that nck-1-mediated INCED against PFP attack is independent of the major MAPK PFP INCED pathways. Proteomics and cell biological and genetic studies further indicate that nck-1 functions with F-actin cytoskeleton modifying genes like arp2/3, erm-1, and dbn-1 and that nck-1/arp2/3 promote pore repair at the membrane surface and protect against PFP attack independent of p38 MAPK. Consistent with these findings, PFP attack causes significant changes in the amount of actin cytoskeletal proteins and in total amounts of F-actin in the target tissue, the intestine. nck-1 mutant animals appear to have lower F-actin levels than wild-type C. elegans. Studies on nck-1 and other F-actin regulating proteins have uncovered a new and important role of this pathway and the actin cytoskeleton in PFP INCED and protecting an intestinal epithelium in vivo against PFP attack. Author SummaryThe mechanism of action for a significant number of bacterial protein toxins is the formation of pores in the membrane of target cells. Host cells contain programmed defenses against such attacks. Here we use the model system of the roundworm Caenorhabditis elegans and crystal proteins produced by Bacillus thuringiensis to demonstrate a new defense pathway mediated by the activity of the NCK-1 protein. Consistent with its known function in mammalian cells, the NCK-1-mediated defense involves various actin-interacting proteins and affects the kinetics of pore repair. This pathway is novel in its independence from MAPK signaling. Furthermore, the NCK-1 activity is found to be generally needed for defense against multiple pore-forming proteins and yet is specific to defense against such proteins but not against other environmental stressors.

microbiology↗

bioRxiv2024

Modular Fluorescent Cholesterol Naphthalimide Probes And Their Application For Cholesterol Trafficking Studies In Cells

Development of fluorescent cholesterol analogs to better understand subcellular cholesterol trafficking is of great interest for cell biology and medicine. Our approach utilizes a bifunctional 1,8-naphthalimide scaffold with a push-pull character, modified on one side with a head group and a linker on the other side connecting it to cholesterol via an ester bond. Through structure-function studies, weve explored how different substituents--linkers and head groups--affect the ability of these fluorescent cholesterol naphthalimide analogs (CNDs) to mimic natural cholesterol behavior at both molecular and cellular levels. We categorized the resulting analogs into three groups: neutral, charged, and those featuring a hydroxyl group. Each compound was assessed for its solvatochromic behavior in organic solvents and model membranes. Extensive all-atom molecular dynamics simulations helped us examine how these analogs perform in model membranes compared to cholesterol. Additionally, we investigated the partitioning of these fluorescent probes in phase-separated giant unilamellar vesicles. We evaluated the uptake and distribution of these probes within mouse fibroblast cells and astrocytes, for their subcellular distributions in lysosomes and compared that to BODIPY-cholesterol, a well-regarded fluorescent cholesterol analog. The internalization efficiency of the fluorescent probes varies in different cell types and is affected mainly by the head groups. Our results demonstrate that the modular design significantly simplifies the creation of fluorescent cholesterol probes bearing distinct spectral, biophysical, and cellular targeting features, which makes it a valuable toolkit for the investigation of subcellular distribution and trafficking of cholesterol and its derivatives.

biochemistry↗

bioRxiv2019

Excitation of Faraday-like body waves in vibrated living earthworms

Biological cells and many living organisms are mostly made of liquids and therefore, by analogy with liquid drops, they should exhibit a range of fundamental nonlinear phenomena such as the onset of standing surface waves. Here, we test four common species of earthworm to demonstrate that vertical vibration of living worms lying horizontally of a flat solid surface results in the onset of subharmonic Faraday-like body waves, which is possible because earthworms have a hydrostatic skeleton with a flexible skin and a liquid-filled body cavity. Our findings are supported by theoretical analysis based on a model of parametrically excited vibrations in liquid-filled elastic cylinders using material parameters of the worms body reported in the literature. The ability to excite nonlinear subharmonic body waves in a living organism could be used to probe, and potentially to control, important biophysical processes such as the propagation of nerve impulses, thereby opening up avenues for addressing biological questions of fundamental impact.

biophysics↗

bioRxiv2023

Integrated high-confidence and high-throughput approaches for quantifying synapse engulfment by oligodendrocyte precursor cells

Oligodendrocyte precursor cells (OPCs) sculpt neural circuits through the phagocytic engulfment of synapses during development and in adulthood. However, precise techniques for analyzing synapse engulfment by OPCs are limited. Here, we describe a two-pronged cell biological approach for quantifying synapse engulfment by OPCs which merges low-and high-throughput methodologies. In the first method, an adeno-associated virus encoding a pH-sensitive, fluorescently-tagged synaptic marker is expressed in neurons in vivo. This construct allows for the differential labeling of presynaptic inputs that are contained outside of and within acidic phagolysosomal compartments. When followed by immunostaining for markers of OPCs and synapses in lightly fixed tissue, this approach enables the quantification of synapses engulfed by around 30-50 OPCs within a given experiment. In the second method, OPCs isolated from dissociated brain tissue are fixed, incubated with fluorescent antibodies against presynaptic proteins, and then analyzed by flow cytometry. This approach enables the quantification of presynaptic material within tens of thousands of OPCs in less than one week. These methods extend beyond the current imaging-based engulfment assays designed to quantify synaptic phagocytosis by brain-resident immune cells, microglia. Through the integration of these methods, the engulfment of synapses by OPCs can be rigorously quantified at both the individual and populational levels. With minor modifications, these approaches can be adapted to study synaptic phagocytosis by numerous glial cell types in the brain.

neuroscience↗

bioRxiv2019

Repertoire of morphable proteins in an organism

All living organisms have evolved to contain a set of proteins with variable physical and chemical properties. Efforts in the field of structural biology have contributed to uncovering the shape and the variability of each component. A set of experimental coordinates for a given protein can be used to define the \"morphness/unmorphness\". Here we show the results of global analysis of more than a thousand E. coli proteins, demonstrating that it would be a comprehensive method of understanding the evolved repertoire in an organism. By collecting \"UnMorphness Factor\" (UMF) determined for each of the proteins, the lowest and the highest boundaries of the experimentally observable structural variation are understood. The distribution of UMFs obtained for an organism is expected to represent how rigid and flexible components are balanced. The present analysis extends to evaluate the growing data from single particle cryo-electron microscopy, providing valuable information on effective interpretation to structural changes of proteins and the supramolecular complexes. The data and the method presented here also conform to FAIR data principles, having potential significance to advance the field of structural and molecular cell biology.

biophysics↗

bioRxiv2019

What is that molecular machine really doing? Automated exploration of alternative transporter mechanisms

Motivated by growing evidence for pathway heterogeneity and alternative functions of molecular machines, we demonstrate a computational approach for investigating two questions: (1) Are there multiple mechanisms (state-space pathways) by which a machine can perform a given function, such as cotransport across a membrane? (2) How can additional functionality, such as proofreading/error-correction, be built into machine function using standard biochemical processes? Answers to these questions will aid both the understanding of molecular-scale cell biology and the design of synthetic machines. Focusing on transport in this initial study, we sample a variety of mechanisms by employing Metropolis Markov chain Monte Carlo. Trial moves adjust transition rates among an automatically generated set of conformational and binding states while maintaining fidelity to thermodynamic principles and a user-supplied fitness/functionality goal. Each accepted move generates a new model. The simulations yield both single and mixed reaction pathways for cotransport in a simple environment with a single substrate along with a driving ion. In a "competitive" environment including an additional decoy substrate, several qualitatively distinct reaction pathways are found which are capable of extremely high discrimination coupled to a leak of the driving ion, akin to proofreading. The array of functional models would be difficult to find by intuition alone in the complex state-spaces of interest. Author summaryMolecular machines, which operate on the nanoscale, are proteins/complexes that perform remarkable tasks such as the selective absorption of nutrients into the cell by transporters. These complex machines are often described using a fairly simple set of states and transitions that may not account for the stochasticity and heterogeneity generally expected at the nanoscale at body temperature. New tools are needed to study the full array of possibilities. This study presents a novel in silico method to systematically generate testable molecular-machine kinetic models and explore alternative mechanisms, applied first to membrane transport proteins. Our initial results suggest these transport machines may contain mechanisms which detoxify the cell of an unwanted toxin, as well as significantly discriminate against the import of the toxin. This novel approach should aid the experimental study of key physiological processes such as renal glucose re-absorption, rational drug design, and potentially the development of synthetic machines.

biophysics↗

bioRxiv2025

Physiological and functional characterization for high-throughput optogenetic skeletal muscle exercise assays

Exercise has long been considered an essential part of human health and longevity. Recent physiological studies have expanded muscles role beyond simply acting as an actuator, revealing muscles exercise-mediated paracrine and endocrine relationships with other organ systems. In vitro engineered skeletal muscle models can address physiological questions about exercise adaptation with the precision of cell biology. Optogenetic tools have enabled a noninvasive approach to stimulating muscle contraction that avoids the potential off-target effects of electrical stimulation techniques. In this article we propose high-throughput culture and optical exercise protocols to generate statistically robust cellular exercise response datasets. We characterize optical rheobase for 2D muscle tissue morphology, finding that optical intensities as low as 5 W mm-2 can trigger contraction. We then analyze bulk RNA sequencing data collected from high throughput, acute exercise protocols and find a rich display of transcriptional behavior that is consistent with experimental observations. The spontaneous contractility of our tissue constructs introduced oxygen diffusion challenges when maintained in a 24 well plate, and our analysis shows divergent myogenic and pathological transcriptional consequences of hypoxia. We believe our techniques provide a practical foundation for conducting future high-precision in vitro exercise studies of skeletal muscle. Translational impactHigh-fidelity, engineered skeletal muscle has potential to elucidate exercise-mediated response pathways at the cell and tissue level. We leverage optogenetic techniques to develop a high-throughput assay that optically stimulates 2D muscle monolayers, avoiding potential cell damage from electrical stimulation. Our culture and exercise protocol generates statistically robust RNA sequencing datasets which reveal myogenic and pathological responses to exercise and in vitro culture conditions, informing practical next steps to cultivate stronger, more physiologically relevant muscle models.

bioengineering↗

bioRxiv2026

TurboID proximity labeling reveals protein interaction networks for germline Argonautes

In the germline, small RNA-mediated gene regulatory pathways are associated with phase separated condensates known as germ granules across species. Germ granules can be challenging to purify based on their biophysical properties, therefore we used an in vivo proximity labeling approach, TurboID, to define the proximal interaction networks of the germ granule-localized Argonaute proteins: CSR-1, PRG-1/Piwi, PPW-2/WAGO-3, WAGO-1 and WAGO-4, and the nuclear-enriched Argonaute HRDE-1 as a comparator, in C. elegans. We found that although CSR-1 and WAGO-4 bind to the same set of 22G- small RNAs and target an overlapping set of germline genes, they possess distinct proximal interaction networks. Displacement of WAGO-4 from the germ granules highlights distinct interactors involved in chromosome biology and points to a potential nuclear role for WAGO-4. PRG-1/Piwi, WAGO-1, and PPW-2/WAGO-3, act in a common pathway and share an interaction network, while HRDE-1 displays a distinct interaction network that highlights its potential to influence splicing, rather than its widely accepted role in histone modification. Performing TurboID on other sRNA pathway factors, including the RdRP RRF-1 led us to uncover a potential nuclear role for this protein, and overlapping our TurboID data with published proximity labeling data enabled us to identify a core set of eleven mostly uncharacterized proteins that play roles in fertility, germ granule organization, and transgenerational epigenetic inheritance. One of these factors, D2005.4, appears to be a heme-binding germ granule-localized protein with a Tetratricopeptide Repeat Domain that interacts with all germ granule AGOs and may play scaffolding or enzymatic roles in germ granules. Collectively, our work highlights the utility of proximity labelling to uncover insights into AGO and sRNA pathway factor function and identify new factors that could be of importance in germ cell biology.

developmental biology↗

bioRxiv2021

Cbp1, a rapidly evolving fungal virulence factor, forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel "binocular" fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a rapidly evolving fungal virulence factor that localizes to the cytosol to trigger host cell lysis. Author SummaryThe members of the Ajellomycetaceae fungal family are human pathogens that are responsible for a rising number of mycoses around the world. Calcium binding protein 1 (Cbp1) is a rapidly evolving virulence factor that is present in the genomes of the Ajellomycetaceae species that lead primarily intracellular lifestyles, including Histoplasma, Paracoccidioides, and Emergomyces but not Blastomyces, which remains largely extracellular during infection. Both Paracoccidioides and Histoplasma Cbp1 homologs are able to cause lysis of macrophages whereas Emergomyces homologs cannot. This result is consistent with Emergomyces africanus natural infection of macrophages, during which the yeast cells can replicate but cannot actively lyse the host cell. Despite divergence of the primary sequence of Histoplasma and Paracoccidioides Cbp1 homologs, their protein structures are remarkably similar and reveal a novel fold. During infection, Cbp1 enters the cytosol of the host macrophage, making it the first known virulence factor from an intracellular human fungal pathogen that localizes to the cytosol of the host cell. We also show that Cbp1 forms a complex with another cytosolic virulence factor, Yps-3. Taken together, these studies significantly advance our understanding of Histoplasma virulence.

microbiology↗

bioRxiv2024

Modified histone peptides uniquely tune the material properties of HP1α condensates.

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1) with DNA contributes to the formation of condensed chromatin states. HP1 localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1 and dictating emergent functions of its condensates, remains only partially understood. Here, we leverage a reductionist system, comprised of modified and unmodified histone H3 peptides, HP1 and DNA to examine the contribution of specific epigenetic marks to phase behavior of HP1. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1 condensates, while peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1 phase separation. In addition, inspired by the decreased ratio of nuclear H3K9me3 to HP1 detected in cells exposed to uniaxial strain, using fluorescence microscopy and rheological approaches we demonstrate that H3K9me3 histone peptides modulate the dynamics and network properties of HP1 condensates in a concentration dependent manner. These data suggest that HP1-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells in response to mechanostimulation. Statement of significanceThe organization of genomic information in eukaryotic cells necessitates compartmentalization into functional domains allowing for the expression of cell identity-specific genes, while repressing genes related to alternative fates. Heterochromatin hosts these transcriptionally silent regions of the genome - which ensure the stability of cell identity -and is characterized by repressive histone marks (H3K9m3) and other specialized proteins (HP1a), recently shown to phase separate with DNA. We show that HP1a forms condensates with DNA which persist in the presence of H3K9me3 peptides. The viscoelastic nature of these condensates depend on H3K9me3:HP1 ratios, which are modulated by mechanical strain in cells. Thus, phase separation may explain the dynamic behavior of heterochromatin in cells, in response to mechanostimulation.

biophysics↗

bioRxiv2022

Dynamics and structural changes of calmodulin upon interaction with its potent antagonist calmidazolium

Calmodulin (CaM) is a eukaryotic multifunctional, calcium-modulated protein that regulates the activity of numerous effector proteins involved in a variety of physiological processes. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Here, we report the structural characterization of CaM:CDZ complexes using combined SAXS, X-ray crystallography, HDX-MS and NMR approaches. Our results provide molecular insights into the CDZ-induced dynamics and structural changes of CaM leading to its inhibition. CDZ-binding induces an open-to-closed conformational change of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from protein targets, despite their distant chemical nature. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. These results open the way to rationally design new CaM-selective drugs. Figure and text for the Table of Contents (ToC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/474921v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@b0c76org.highwire.dtl.DTLVardef@15f462forg.highwire.dtl.DTLVardef@1f8e57forg.highwire.dtl.DTLVardef@1a33575_HPS_FORMAT_FIGEXP M_FIG C_FIG Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results open the way to rationally design new and more selective inhibitors of calmodulin.

biophysics↗

bioRxiv2022

The growth rate of DNA condensate droplets increases with the size of participating subunits

Liquid-liquid phase separation (LLPS) is a common phenomenon underlying the formation of dynamic membraneless organelles in biological cells, which are emerging as major players in controlling cellular functions and health. The bottom-up synthesis of biomolecular liquid systems with simple constituents, like nucleic acids and peptides, is useful to understand LLPS in nature as well as to develop programmable means to build new amorphous materials with properties matching or surpassing those observed in natural condensates. In particular, understanding which parameters determine condensate growth kinetics is essential for the synthesis of condensates with the capacity for active, dynamic behaviors. Here we use DNA nanotechnology to study artificial liquid condensates through programmable star-shaped subunits, focusing on the effects of changing subunit size. First, we show that LLPS is achieved in a six-fold range of subunit size. Second, we demonstrate that the rate of growth of condensate droplets scales with subunit size. Our investigation is supported by a general model that describes how coarsening and coalescence are expected to scale with subunit size under ideal assumptions. Beyond suggesting a route toward achieving control of LLPS kinetics via design of subunit size in synthetic liquids, our work suggests that particle size may be a key parameter in biological condensation processes.

bioengineering↗

bioRxiv2024

Aggregation and disaggregation of red blood cells: depletion versus bridging

The aggregation of red blood cells (RBCs) is a complex phenomenon that strongly impacts blood flow and tissue perfusion. Despite extensive research for more than 50 years, physical mechanisms that govern RBC aggregation are still under debate. Two proposed mechanisms are based on bridging and depletion interactions between RBCs due to the presence of macromolecules in blood plasma. The bridging hypothesis assumes the formation of bonds between RBCs through adsorbing macromolecules, while the depletion mechanism results from the exclusion of macromolecules from the inter-cellular space, leading to effective attraction. Existing experimental studies generally cannot differentiate between these two aggregation mechanisms, though several recent investigations suggest concurrent involvement of the both mechanisms. We explore dynamic aggregation and disaggregation of two RBCs using three simulation models: a potential-based model mimicking depletion interactions, a bridging model with immobile bonds, and a new bridging model with mobile bonds which can slide along RBC membranes. Simulation results indicate that dynamic aggregation of RBCs primarily arises from depletion interactions, while disaggregation of RBCs involves both mechanisms. The bridging model with mobile bonds reproduces well the corresponding experimental data, offering insights into the interplay between bridging and depletion interactions and providing a framework for studying similar interactions between other biological cells.

biophysics↗

bioRxiv2022

Noise source importance in linear stochastic models of biological systems that grow, shrink, wander, or persist

While noise is an important factor in biology, biological processes often involve multiple noise sources, whose relative importance can be unclear. Here we develop tools that quantify the importance of noise sources in a network based on their contributions to variability in a quantity of interest. We generalize the edge importance measures proposed by Schmidt and Thomas [1] for first-order reaction networks whose steady-state variance is a linear combination of variance produced by each directed edge. We show that the same additive property extends to a general family of stochastic processes subject to a set of linearity assumptions, whether in discrete or continuous state or time. Our analysis applies to both expanding and contracting populations, as well as populations obeying a martingale ("wandering") at long times. We show that the original Schmidt-Thomas edge importance measure is a special case of our more general measure, and is recovered when the model satisfies a conservation constraint ("persists"). In the growing and wandering cases we show that the choice of observables (measurements) used to monitor the process does not influence which noise sources are important at long times. In contrast, in the shrinking or persisting case, which noise sources are important depends on what is measured. We also generalize our measures to admit models with affine moment update equations, which admit additional limiting scenarios, and arise naturally after linearization. We illustrate our results using examples from cell biology and ecology: (i) a model for the dynamics of the inositol trisphospate receptor, (ii) a model for an endangered population of white-tailed eagles, and (iii) a model for wood frog dispersal. Author summaryBiological processes are frequently subject to an ensemble of independent noise sources. Noise sources produce fluctuations that propagate through the system, driving fluctuations in quantities of interest such as population size or ion channel configuration. We introduce a measure that quantifies how much variability each noise source contributes to any given quantity of interest. Using these methods, we identify which binding events contribute significantly to fluctuations in the state of a molecular signalling channel, which life history events contribute the most variability to an eagle population before and after a successful conservation effort rescued the population from the brink of extinction, and which dispersal events, at what times, matter most to variability in the recolonization of a series of ponds by wood frogs after a drought.

ecology↗

bioRxiv2019

Kinetochore capture by spindle microtubules: why fission yeast may prefer pivoting to search-and-capture

The mechanism by which microtubules find kinetochores during spindle formation is a key question in cell biology. Previous experimental studies have shown that although search-and-capture of kinetochores by dynamic microtubules is a dominant mechanism in many organisms, several other capture mechanisms are also possible. One such mechanism reported in Schizosaccharomyces pombe shows that microtubules can exhibit a prolonged pause between growth and shrinkage. During the pause, the microtubules pivoted at the spindle pole body search for the kinetochores by performing an angular diffusion. Is the latter mechanism purely accidental, or could there be any physical advantage underlying its selection? To compare the efficiency of these two mechanisms, we numerically study distinct models and compute the timescales of kinetochore capture as a function of microtubule number N. We find that the capture timescales have non-trivial dependences on microtubule number, and one mechanism may be preferred over the other depending on this number. While for small N (as in fission yeast), the typical capture times due to rotational diffusion are lesser than those for search-and-capture, the situation is reversed beyond a certain N. The capture times for rotational diffusion tend to saturate due to geometrical constraints, while those for search-and-capture reduce monotonically with increasing N making it physically more efficient. The results provide a rationale for the common occurrence of classic search-and-capture process in many eukaryotes which have few hundreds of dynamic microtubules, as well as justify exceptions in cells with fewer microtubules.

biophysics↗

bioRxiv2023

Geckopy 3.0: enzyme constraints, thermodynamics constraints and omics integration in python

GEnome-scale Metabolic (GEM) models are knowledge bases of the reactions and metabolites of a particular organism. These GEM models allow for the simulation of the metabolism - e.g. calculating growth and production yields - based on the stoichiometry, reaction directionality and uptake rates of the metabolic network. Over the years, several extensions have been added to take into account other actors in metabolism, going beyond pure stoichiometry. One such extension is enzyme-constraint models, which enable the integration of kinetic data and proteomics data into GEM models. Given its relatively recent formulation, there are still challenges in standardization and data reconciliation between the model and the experimental measurements. In this work, we present geckopy 3.0 (Genome-scale model Enzyme Constraints, using Kinetics and Omics in python), an actualization from scratch of the previous python implementation of the same name. This update tackles the aforementioned challenges, in an effort to reach maturity in enzyme-constraint modeling. With the new geckopy, proteins are typed in the SBML document, taking advantage of the SBML Groups extension, in compliance with community standards. Additionally, a suite of relaxation algorithms - in the form of linear and mixed-integer linear programming problems - has been added to facilitate reconciliation of raw proteomics data with the metabolic model. Several functionalities to integrate experimental data were implemented, including an interface layer with pytfa for the usage of thermodynamics and metabolomics constraints. Finally, the relaxation algorithms were benchmarked against public proteomics datasets in Escherichia coli for different conditions, revealing targets for improving the enzyme constrained model and/or the proteomics pipeline. IMPORTANCEThe metabolism of biological cells is an intricate network of reactions that interconvert chemical compounds, gathering energy and using that energy to grow. The static analysis of these metabolic networks can be turned into a computational model which is able to efficiently output the distribution of fluxes in the network. With the inclusion of enzymes in the network, we can also interpret the role and concentrations of the metabolic proteins. However, the models and the experimental data often clash, resulting in a network that cannot grow. Here, we tackle this situation with a suite of relaxations algorithms in a package called geckopy. Additionally, to ensure that enzyme-constraint models follow the community standards, a format for the proteins is postulated. Geckopy also integrates with other software to allow for adding thermodynamic and metabolomic constraints. We hope that the package and algorithms presented here will serve useful for the constraint-based modeling community.

systems biology↗

bioRxiv2026

Atg8 orchestrates stress-responsive chromatin programs across immunity and metabolism

Organisms must coordinate transcriptional responses to immune and metabolic stress, often within the same tissue. In Drosophila and mammals, adipose tissue integrates these signals by mounting antimicrobial defense during acute infection and remodeling lipid metabolism under chronic nutrient surplus. How one cell-biological system supports both functions, and through what molecular machinery, remains incompletely understood. Atg8/LC3, classically defined by canonical autophagy, has emerging non-canonical roles in nuclear gene regulation, raising the possibility that it contributes to stress-coordinated transcription beyond cargo turnover. Using unbiased CUT&RUN in adult Drosophila nuclei, we find that endogenous Atg8 exhibits broad chromatin occupancy at immune, metabolic, and autophagy loci, and accumulates in nuclei under prolonged high-sugar diet (HSD) and acute Gram-positive infection. We identify two conserved Atg8-interacting motifs (AIMs) within the Rel homology domain of NF-{kappa}B/Dif. Flies carrying CRISPR-engineered AIM-mutant Dif are highly susceptible to both infection and chronic HSD, establishing a physiological requirement for intact Dif AIMs. AIM-mutant Dif shows impaired infection-induced nuclear accumulation, suggesting that Atg8 contributes to both Dif cytoplasmic-to-nuclear shuttling and nuclear function. Unbiased comparison of Atg8 chromatin occupancy across HSD and infection further reveals shared and divergent motif grammar, positioning Atg8 as a stress-responsive chromatin cofactor for immune and metabolic transcription. Together, these findings expand the functional landscape of Atg8/LC3 beyond canonical autophagy and reveal that autophagy machinery contributes to stress-specific transcriptional complex assembly. AIM/LIR-mediated interactions, exemplified by Dif, represent one such interface, while additional mechanisms likely underlie Atg8s broader chromatin engagement at loci enriched for transcription factor motifs whose cognate factors lack known AIM/LIRs. We propose that Atg8/LC3-mediated coordination of immune and metabolic transcription is a general principle by which cells integrate diverse stress signals, with implications for obesity, chronic inflammation, and other disease states in which immune and metabolic dysregulation converge. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/727304v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@15c056dorg.highwire.dtl.DTLVardef@68493dorg.highwire.dtl.DTLVardef@a07c6corg.highwire.dtl.DTLVardef@4896c6_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIStress drives Atg8 into nuclei, where it occupies immune and metabolic chromatin. C_LIO_LITwo conserved AIMs in NF-{kappa}B/Dif bind Atg8 and enable Dif nuclear entry. C_LIO_LIAIM-mutant Dif flies are highly susceptible to infection and chronic high-sugar diet. C_LIO_LIAtg8 occupies stress-related motifs on prolonged HSD and acute infection. C_LI

genomics↗