Biology subjects

Dinner, A. R.

Publications and source records attributed to Dinner, A. R..

18 recordsLinked to original sources

bioRxiv2025

Mapping the diverse topologies of protein-protein interaction fitness landscapes

De novo binder discovery is unpredictable and inefficient due to a lack of quantitative understanding of protein-protein interaction (PPI) sequence-function landscapes. Here, we use our PANCS-Binder technology to perform >1,300 independent selections of various library sizes and compositions of a randomized small protein to identify binders to a panel of 96 distinct target proteins. For successful selections, we discovered reproducible fitness landscapes that group into a few, target-specific, clusters. Each cluster defines a minimal binding motif whose frequency is inversely proportional to the number of specified amino acids ([~]2-8) and determines selection success, which is quantifiable by the density of binders to the target within a theoretical sequence space. We leverage these data to develop a supervised contrastive learning approach that discriminates binders from non-binders and demonstrates generalization beyond a threshold amount of data. Together, this framework renders PPI landscapes measurable and predictive, accelerating de novo binder discovery and optimization.

synthetic biology↗

bioRxiv2025

Tau-Driven Coordination of Microtubule-Actin Crosstalk in Cell-Sized Vesicles

The coordination of microtubules (MTs) and actin filaments is essential for cytoskeletal organization, yet the factors that affect their integration remain unclear. Here, we reconstitute cytoskeletal networks in giant unilamellar vesicles to characterize MT-actin crosstalk mediated by tau, a microtubule-associated protein. We show that tau promotes the organization of MTs into diverse architectures, including bundles, clusters, and networks, depending on its concentration and vesicle size. In vitro assays confirm that while tau binds and bundles MTs, it does not directly bundle actin. However, tau facilitates MT-actin colocalization in the presence of actin crosslinkers with distinct properties. Fascin, which forms rigid actin bundles, significantly enhances MT-actin colocalization with tau, whereas -actinin, which forms flexible actin bundles, induces colocalization in vesicles but not in bulk conditions. By combining cellular reconstitution and coarse-grained simulations of composite network assembly in both vesicles and bulk conditions, our findings reveal how tau-mediated cytoskeletal integration is governed by bundle mechanics and spatial confinement, providing insights into cytoskeletal organization within reconstituted synthetic cell-like systems.

biophysics↗

bioRxiv2024

Fishnet mesh of centrin-Sfi1 drives ultrafast calcium-activated contraction of the giant cell Spirostomum ambiguum

Spirostomum is a giant unicellular ciliate that contracts to a quarter of its body length in less than five milliseconds, achieving an order of magnitude higher fractional shortening rate than actomyosin-based systems. This ultrafast contraction is powered by myonemes, calcium-activated protein networks at the cortex whose biochemical mechanism remain unclear. We quantify changes in cortical microtubules, membrane ruffles, and the fishnet-like myoneme mesh during contraction, and develop multiscale models that connect local myoneme shortening to whole-cell shape change. Centrin and an Sfi1 homolog co-localize with the myoneme by immunofluorescence and localize to the myoneme by immunogold electron microscopy. Coarse-grained mesh simulations reproduce the measured deformations and show that fishnet geometry, together with volume conservation, leads to uniform contraction. Finally, we reconstitute a Spirostomum centrin-Sfi1 repeat complex in vitro and measure calcium-dependent compaction and self-association, supporting a molecular basis for myoneme contractility. Together, these results support a multiscale model in which calcium-responsive centrin- Sfi1 structures are the central contractile element in Spirostomum and suggest design principles for fast, calcium-triggered, chemomechanical contractile networks that operate without actomyosin or ATP. SIGNIFICANCE STATEMENTMany cells change shape using actomyosin, but some protists contract using calcium-activated protein networks called myonemes. We combine quantitative imaging, electron microscopy, multiscale modeling, and in vitro reconstitution to link molecular-scale mechanisms to the millisecond shortening of the giant ciliate Spirostomum. Centrin and an Sfi1 homolog co-localize in a fishnet-like cortical mesh, and simulations show that this geometry can reproduce the observed whole-cell shape change under volume conservation. Purified centrin-Sfi1 complexes undergo calcium-dependent compaction and self-association, supporting a protein-scale switch that can drive myoneme contraction. These results connect calcium signaling to whole-cell mechanics and suggest principles for designing fast, ATP-independent bioin-spired actuators and synthetic cellular machinery.

cell biology↗

bioRxiv2024

Analysis of the dynamics of a complex, multipathway reaction: Insulin dimer dissociation

The protein hormone insulin forms a homodimer that must dissociate to bind to its receptor. Understanding the kinetics and mechanism of dissociation is essential for rational design of therapeutic analogs. In addition to its physiological importance, this dissociation process serves as a paradigm for coupled (un)folding and (un)binding. Based on previous free energy simulations, insulin dissociation is thought to involve multiple pathways with comparable free energy barriers. Here, we analyze the mechanism of insulin dimer dissociation using a recently developed computational framework for estimating kinetic statistics from short-trajectory data. These statistics indicate that the likelihood of dissociation (the committor) closely tracks the decrease in the number of (native and nonnative) intermonomer contacts and the increase in the number of water contacts at the dimer interface; the transition state with equal likelihood of association and dissociation corresponds to an encounter complex with relatively few native contacts and many nonnative contacts. We identify four pathways out of the dimer state and quantify their contributions to the rate, as well as their exchange, by computing reactive fluxes. We show that both the pathways and their extents of exchange can be understood in terms of rotations around three axes of the dimer structure. Our results provide insights into the kinetics of insulin analogues and, more generally, how to characterize complex, multipathway processes.

biophysics↗

bioRxiv2023

Pervasive, conserved secondary structure in highly charged protein regions

Understanding how protein sequences confer function remains a defining challenge in molecular biology. Two approaches have yielded enormous insight yet are often pursued separately: structure-based, where sequence-encoded structures mediate function, and disorder-based, where sequences dictate physicochemical and dynamical properties which determine function in the absence of stable structure. Here we study highly charged protein regions (>40% charged residues), which are routinely presumed to be disordered. Using recent advances in structure prediction and experimental structures, we show that roughly 40% of these regions form well-structured helices. Features often used to predict disorder--high charge density, low hydrophobicity, low sequence complexity, and evolutionarily varying length--are also compatible with solvated, variable-length helices. We show that a simple composition classifier predicts the existence of structure far better than well-established heuristics based on charge and hydropathy. We show that helical structure is more prevalent than previously appreciated in highly charged regions of diverse proteomes and characterize the conservation of highly charged regions. Our results underscore the importance of integrating, rather than choosing between, structure- and disorder-based approaches.

bioinformatics↗

bioRxiv2022

Dynamics of activation in the voltage-sensing domain of Ci-VSP

The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) is a membrane protein containing a voltagesensing domain (VSD) that is homologous to VSDs from voltage-gated ion channels responsible for cellular excitability. Two crystal structures of Ci-VSD in putative resting and active conformations suggest a helical-screw voltage sensing mechanism in which the S4 helix translocates and rotates to enable exchange of salt-bridge partners. By combining extensive molecular dynamics simulations with a computational framework based on dynamical operators, we elucidate the microscopic mechanism of the resting-active transition at physiological membrane potential. Sparse regression reveals a small set of coordinates that distinguish intermediates hidden from electrophysiological measurements. The intermediates arise from a noncanonical helical-screw mechanism in which translocation, rotation, and side-chain movement of the S4 helix are only loosely coupled. These results provide new insights into existing experimental and computational findings on voltage sensing and suggest ways of further probing its mechanism.

biophysics↗

bioRxiv2022

Volumetric microscopy of CD9 and CD63 reveals distinct subpopulations and novel structures of extracellular vesicle in situ in triple negative breast cancer cells

Secreted extracellular vesicles (EVs) are now known to play multifaceted roles in biological processes such as immune responses and cancer. The two primary classes of EVs are defined in terms of their origins: exosomes are derived from the endosomal pathway while microvesicles (ectosomes) bud from the cell membrane. However, it remains unclear whether the contents, sizes, and localizations of subpopulations of EVs can be used to associate them with the two primary classes. Here, we use confocal microscopy and high-resolution volumetric imaging to study intracellular localization of the EV markers CD9 and CD63 prior to EV export from cells. We find significantly different spatial expression of CD9 and CD63. CD9 is primarily localized in microvesicles, while CD63 is detected exclusively in exosomes. We also observe structures in which CD63 forms a shell that encapsulates CD9 and interpret them to be multi-vesicular bodies. The morphology and location within the endoplasmic reticulum of these shell-like structures are consistent with a role in differential sorting and export of exosomes and microvesicles. Our in situ imaging allows unambiguous identification and tracking of EVs from their points of origin to cell export, and suggest that CD9 and CD63 can be used as biomarkers to differentiate subpopulations of EVs.

biophysics↗

bioRxiv2022

A unified model for the dynamics of ATP-independent ultrafast contraction

In nature, several ciliated protists possess the remarkable ability to execute ultrafast motions using protein assemblies called myonemes, which contract in response to Ca2+ ions. Existing theories, such as actomyosin contractility and macroscopic biomechanical latches, do not adequately describe these systems, necessitating new models to understand their mechanisms. In this study, we image and quantitatively analyze the contractile kinematics observed in two ciliated protists (Vorticella sp and Spirostomum sp), and, based on the mechanochemistry of these organisms, we propose a minimal mathematical model that reproduces our observations as well as those published previously. Analyzing the model reveals three distinct dynamic regimes, differentiated by the rate of chemical driving and the importance of inertia. We characterize their unique scaling behaviors and kinematic signatures. Besides providing insights into Ca2+-powered myoneme contraction in protists, our work may also inform the rational design of ultrafast bioengineered systems such as active synthetic cells.

biophysics↗

bioRxiv2021

Actin filament alignment causes mechanical hysteresis in cross-linked networks

Cells dynamically control their material properties through remodeling of the actin cytoskeleton, an assembly of cross-linked networks and bundles formed from the biopolymer actin. We recently found that cross-linked networks of actin filaments reconstituted in vitro can exhibit adaptive behavior and thus serve as a model system to understand the underlying mechanisms of mechanical adaptation of the cytoskeleton. In these networks, training, in the form of applied shear stress, can induce asymmetry in the nonlinear elasticity. Here, we explore control over this mechanical hysteresis by tuning the concentration and mechanical properties of cross-linking proteins in both experimental and simulated networks. We find that this effect depends on two conditions: the initial network must exhibit nonlinear strain stiffening, and filaments in the network must be able to reorient during training. Hysteresis depends strongly and non-monotonically on cross-linker concentration, with a peak at moderate concentrations. In contrast, at low concentrations, where the network does not strain stiffen, or at high concentrations, where filaments are less able to rearrange, there is little response to training. Additionally, we investigate the effect of changing cross-linker properties and find that longer or more flexible cross-linkers enhance hysteresis. Remarkably plotting hysteresis against alignment after training yields a single curve regardless of the physical properties or concentration of the cross-linkers.

biophysics↗

bioRxiv2020

Kinetic model of small RNA-mediated regulation suggests that a small RNA can regulate co-transcriptionally

Small RNAs (sRNAs) are important regulators of gene expression in bacteria, particularly during stress responses. Many genetically and biochemically well characterized sRNAs regulate gene expression post-transcriptionally, by affecting translation and degradation of the target mRNA after they bind to their targets through base pairing. However, how regulation at each of these levels quantitatively contributes to the overall efficacy of sRNA-mediated regulation is not well understood. Here we present a general approach combining imaging and mathematical modeling to determine kinetic parameters at different levels of sRNA-mediated gene regulation. Unexpectedly, our data reveal that certain previously characterized sRNAs are able to regulate some targets co-transcriptionally, rather strictly post-transcriptionally, and suggest that sRNA-mediated regulation can occur early in the mRNAs lifetime, perhaps as soon as the sRNA binding site is transcribed. In addition, our data suggest several important kinetic steps that may determine the efficiency and differential regulation of multiple mRNA targets by an sRNA. Particularly, binding of sRNA to the target mRNA is likely the rate-limiting step and may dictate the regulation hierarchy observed within an sRNA regulon.

biophysics↗

bioRxiv2020

Insulin dissociates by diverse mechanisms of coupled unfolding and unbinding

The protein hormone insulin exists in various oligomeric forms, and a key step in binding its cellular receptor is dissociation of the dimer. This dissociation process and its corresponding association process have come to serve as a paradigms of coupled (un)folding and (un)binding more generally. Despite its fundamental and practical importance, the mechanism of insulin dimer dissociation remains poorly understood. Here, we use molecular dynamics simulations, leveraging recent developments in umbrella sampling, to characterize the energetic and structural features of dissociation in unprecedented detail. We find that the dissociation is inherently multipathway with limiting behaviors corresponding to conformational selection and induced fit, the two prototypical mechanisms of coupled folding and binding. Along one limiting path, the dissociation leads to detachment of the C-terminal segment of the insulin B chain from the protein core, a feature believed to be essential for receptor binding. We simulate IR spectroscopy experiments to aid in interpreting current experiments and identify sites where isotopic labeling can be most effective for distinguishing the contributions of the limiting mechanisms.

biophysics↗

bioRxiv2019

Bayesian Modeling Reveals Ultrasensitivity Underlying Metabolic Compensation in the Cyanobacterial Circadian Clock

Mathematical models can enable a predictive understanding of mechanism in cell biology by quantitatively describing complex networks of interactions, but such models are often poorly constrained by available data. Owing to its relative biochemical simplicity, the core circadian oscillator in Synechococcus elongatus has become a prototypical system for studying how collective dynamics emerge from molecular interactions. The oscillator consists of only three proteins, KaiA, KaiB, and KaiC, and near-24-h cycles of KaiC phosphorylation can be reconstituted in vitro. Here, we formulate a molecularly-detailed but mechanistically agnostic model of the KaiA-KaiC subsystem and fit it directly to experimental data within a Bayesian parameter estimation framework. Analysis of the fits consistently reveals an ultrasensitive response for KaiC phosphorylation as a function of KaiA concentration, which we confirm experimentally. This ultrasensitivity primarily results from the differential affinity of KaiA for competing nucleotide-bound states of KaiC. We argue that the ultrasensitive stimulus-response relation is critical to metabolic compensation by suppressing premature phosphorylation at nighttime. SynopsisThis study takes a data-driven kinetic modeling approach to characterizing the interaction between KaiA and KaiC in the cyanobacterial circadian oscillator and understanding how the oscillator responds to changes in cellular metabolic conditions. O_LIAn extensive dataset of KaiC autophosphorylation measurements was gathered and fit to a detailed yet mechanistically agnostic kinetic model within a Bayesian parameter estimation framework. C_LIO_LIKaiA concentration tunes the sensitivity of KaiC autophosphorylation and the period of the full oscillator to %ATP. C_LIO_LIThe model reveals an ultrasensitive dependence of KaiC phosphorylation on KaiA concentration as a result of differential KaiA binding affinity to ADP- vs. ATP-bound KaiC. C_LIO_LIUltrasensitivity in KaiC phosphorylation contributes to metabolic compensation by suppressing premature phosphorylation at nighttime. C_LI

systems biology↗

bioRxiv2018

Mechanical and kinetic factors drive sorting of F-actin crosslinkers on bundles

In cells, actin binding proteins (ABPs) sort to different regions in order to establish F-actin networks with diverse functions, including filopodia used for cell migration, or contractile rings required for cell division. Recent experimental work uncovered a passive mechanism that may facilitate spatial localization of ABPs: binding of a short crosslinker protein to two actin filaments promotes the binding of other short crosslinkers and inhibits the binding of longer crosslinkers (and vice versa). We hypothesize this sorting arises because F-actin is semiflexible and cannot bend over short distances. We develop a mathematical theory and a kinetic Monte Carlo simulation encompassing the most important physical parameters for this process, and use simulations of a coarse-grained but molecularly explicit model to characterize and test our predictions about the interplay of mechanical and kinetic parameters. Our theory and data predict an explicit dependence of crosslinker separation on bundle polymerization rate. We perform experiments that confirm a dependence on polymerization rate, but in an unanticipated non-monotonic manner. We use simulations to show that this non-monotonic behavior can arise in situations where crosslinkers have equal bundling affinity at equilibrium, but differing microscopic binding rates to filaments. This dependence of sorting on actin polymerization rate is a non-equilibrium effect, qualitatively similar to non-equilibrium domain formation in materials growth. Thus our results reveal an avenue by which cells can organize molecular material to drive biological processes, and can also guide the choice and design of crosslinkers for engineered protein-based materials.

biophysics↗

bioRxiv2018

Transient intracellular acidification regulates the core transcriptional heat shock response

Heat shock induces a conserved transcriptional program regulated by heat shock factor 1 (Hsf1) in eukaryotic cells. Activation of this heat-shock response is triggered by heat-induced misfolding of newly synthesized polypeptides, and so has been thought to depend on ongoing protein synthesis. Here, using the the budding yeast Saccharomyces cerevisiae, we report the discovery that Hsf1 can be robustly activated when protein synthesis is inhibited, so long as cells undergo cytosolic acidification. Heat shock has long been known to cause transient intracellular acidification which, for reasons which have remained unclear, is associated with increased stress resistance in eukaryotes. We demonstrate that acidification is required for heat shock response induction in translationally inhibited cells, and specifically affects Hsf1 activation. Physiological heat-triggered acidification also increases population fitness and promotes cell cycle reentry following heat shock. Our results uncover a previously unknown adaptive dimension of the well-studied eukaryotic heat shock response.

cell biology↗

bioRxiv2018

Tuning molecular motor transport through cytoskeletal filament network organization

The interaction of motor proteins with intracellular filaments is required for transport processes and force generation in cells. Within a cell, crosslinking proteins organize cytoskeletal filaments both temporally and spatially to create dynamic, and structurally diverse networks. The architecture of these networks changes both the mechanics as well as the transport dynamics; however, the effects on transport are less well understood. Here, we compare the transport dynamics of myosin II motor proteins moving on model cytoskeletal networks created by common crosslinking proteins. We observe that motor dynamics change predictably based on the microstructure of the underlying networks and discuss how this can be utilized by cells to achieve specific transport goals.

biophysics↗

bioRxiv2017

Filament Rigidity And Connectivity Tune The Deformation Modes Of Active Biopolymer Networks

Molecular motors embedded within collections of actin and microtubule filaments underlie the dynamic behaviors of cytoskeletal assemblies. Understanding the physics of such motor-filament materials is critical to developing a physical model of the cytoskeleton and the design of biomimetic active materials. Here, we demonstrate through experiments and simulations that the rigidity and connectivity of filaments in active biopolymer networks regulates the anisotropy and the length scale of the underlying deformations, yielding materials with varying contractility. Semi-flexible filaments that can be compressed and bent by motor stresses undergo deformations that are predominantly biaxial. By contrast, rigid filament bundles contract via actomyosin sliding deformations that are predominantly uniaxial. Networks dominated by filament buckling are robustly contractile under a wide range of connectivities, while networks dominated by actomyosin sliding can be tuned from contractile to extensile through reduced connectivity via cross-linking. These results identify physical parameters that control the forces generated within motor-filament arrays, and provide insight into the self-organization and mechanics of cytoskeletal assemblies.

biophysics↗

bioRxiv2017

Bootstrapping and Empirical Bayes Methods Improve Rhythm Detection in Sparsely Sampled Data

MotivationThere is much interest in using genome-wide expression time series to identify circadian genes. However, the cost and effort of such measurements often limits data collection. Consequently, it is difficult to assess the experimental uncertainty in the measurements and, in turn, to detect periodic patterns with statistical confidence.\n\nResultsWe show that parametric bootstrapping and empirical Bayes methods for variance shrinkage can improve rhythm detection in genome-wide expression time series. We demonstrate these approaches by building on the empirical JTK_CYCLE method (eJTK) to formulate a method that we term BooteJTK. Our procedure rapidly and accurately detects cycling time series by combining information about measurement uncertainty with information about the rank order of the time series values. We exploit a publicly available genome-wide dataset with high time resolution to show that BooteJTK provides more consistent rhythm detection thanexisting methods at typical sampling frequencies. Then, we apply BooteJTK to genome-wide expression time series from multiple tissues and show that it reveals biologically sensible tissue relationships that eJTK misses.\n\nAvailabilityBootstrap eJTK (BooteJTK) is implemented in Python and is freely available on GitHub at https://github.com/alanlhutchison/BooteJTK.

bioinformatics↗

bioRxiv2017

Correcting for Dependent P-values in Rhythm Detection

There is much interest in using genome-wide expression time series to identify circadian genes. Several methods have been developed to test for rhythmicity in sparsely sampled time series typical of such measurements. Because these methods are statistical in nature, they rely on estimating the probabilities that patterns arise by chance (i.e., p-values). Here we show that leading methods implicitly make inappropriate assumptions of independence when estimating p-values. We show how to correct for the dependence to obtain accurate estimates for statistical significance during rhythm detection.

bioinformatics↗