- by Tisdale, G.Intron length is a fascinating example of form without function. The vast majority of intronic space within genomes remains without a provided utility. It often fascinates us to find introns performing any function at all, establishing an attention bias against the vast lacking of utility of the remaining intergenic space. In an attempt to better understand the greater breadth of intronic length, I investigate here what I term The Kinetic Intron Hypothesis. This hypothesis investigates hypothetical dynamics of intron RNA […]
- A Modular Framework for Automated Segmentation and Analysis of AFM Imaging of Chromatin Organizationby Sorensen, E. W., Pangeni, S., Merino-Urteaga, R., Murray, P. J., Rudnizky, S., Liao, T.-W., Rashid, F., Hwang, J., Yamadi, M., Feng, X. A., Zähringer, J., Gu, S., Davidson, I. F., Caccianini, L., Osorio-Valeriano, M., Farnung, L., Vos, S., Peters, J.-M., Berger, J. M., Wu, C., Hatzakis, N. S., Kirkegaard, J. B., Ha, T.Chromatin organization underlies essential genome functions, but its nanoscale organization remains challenging to capture and quantify with precision. Atomic force microscopy (AFM) offers direct structural readouts of DNA and chromatin, yet translating these rich images into reproducible biological metrics has been limited by the lack of standardized, scalable analysis tools. Here we present DNAsight, an automated analysis framework that integrates machine learning (ML)-based segmentation with modular, base-pair-calibrated quantification of DNA spatial organization, looping, nucleosome spacing, and protein clustering. Applied across […]
- by Smith, E. R., Gelder, K. L., Hunter-Craig, L., Bose, D. A., Craggs, T. D., Twelvetrees, A. E.Fluorescence resonance energy transfer (FRET) is the highly distance dependent (3-10 nm) transfer of energy from a donor to an acceptor fluorophore, with transfer efficiency inversely proportional to the distance between the fluorophores. Consequently FRET serves as a powerful spectroscopic ruler for probing molecular interactions. Whilst cell based FRET assays report bulk relative changes in FRET efficiency in a population, single molecule FRET (smFRET) is capable of deconvoluting these population averages into distinct structural states. However, the lack of universal […]
- by Zhang, Z., Hong, W., Wu, Y., Dey, A., Shevchuk, A., Klenerman, D.Oblique plane microscopy (OPM) is a light sheet microscopy technique that uses a single high numerical aperture (NA) objective for both illuminating the sample and collecting emission fluorescence from a tilted plane within the specimen. OPM has become indispensable in biological and biomedical research, providing rapid, high-resolution volumetric fluorescence imaging of live cells and tissues while minimising phototoxicity and photobleaching. It also overcomes the sample mounting challenges associated with conventional light sheet microscopes that require two orthogonally placed objectives. However, […]
- by Wu, X., Swanger, S. A., Meier, L. E. B., Dennison, C. L., Weinberg, S. H., Poelzing, S., Gourdie, R. G.Electrical communication between excitable cells depends on both direct gap junction (GJ) currents and field mediated ephaptic interactions, but their relative contributions have remained difficult to quantify experimentally, limiting mechanistic insight into arrhythmia and other disorders of bioelectric signaling in excitable tissues. Building on the concept of a nanoscale, sodium channel rich perinexus at the cardiac intercalated disc, we developed a Single on Paired (SoP) preparation in which whole cell sodium current is recorded from one adult ventricular myocyte that […]
- by Brownd, M., Khodadadi, E., Moradi, M.Peptidase-containing ATP-binding cassette transporters (PCATs) couple ATP hydrolysis with proteolytic processing and export of cargo peptides across cellular membranes. Despite their importance in bacterial secretion systems, the molecular determinants governing nucleotide binding and stabilization in PCAT transporters remain incompletely understood. In particular, recent experimental observations suggest that PCAT1 may display altered nucleotide preferences compared with canonical ABC transporters. Here, we employed microsecond-scale all-atom molecular dynamics simulations combined with free energy perturbation (FEP) calculations to characterize nucleotide binding, protein stability, and […]
- by Brownd, M., Chaturvedi, P., Fakharzadeh, A., Moradi, M.Mitochondrial localization peptides (MLPs) play a critical role in directing proteins to mitochondria, yet how subtle sequence variations influence their conformational behavior remains poorly understood. Here, we investigate the conformational dynamics of a 15-residue MLP derived from the androgen receptor, together with a comprehensive panel of single-residue variants generated by systematic substitution at the second position. Across all variants, the peptide remains intrinsically disordered, exhibiting broad conformational heterogeneity and no stable folded state. Global measures of compactness show that single-residue […]
- by Brownd, M., Sauve, S., Woods, H., Moradi, M.Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are are a family of voltage-gated, cyclic-nucleotide modulated Na+/K+ channels that regulate spontaneous rhythmic electrical activity in both the heart and the brain. Understanding differences in the responsiveness to cyclic adenosine monophosphate (cAMP) modulation between HCN isoforms would offer insight into the specific binding interactions that drive channel activation. Using all-atom molecular dynamics (MD) simulations and the free-energy perturbation (FEP) approach, we determined the absolute binding free energy of cAMP to the the cyclic-nucleotide-binding domain […]
- by Ackermann, B. E., Hall, E., Mariscal, V. T., Clark, A., Corbett, K. D., Carlin, A., Guseman, A.The Oscillatoria agardhii agglutinin (OAA) lectin interacts with N-glycans through a pentamannose core shared among all high mannose N-glycans (HMGs). Because HMGs only differ by number of mannose sugars, there is a scarcity of tools sensitive enough to resolve each specific HMG structure in their biological context. Here, we investigate the sequence space of OAA to tune the binding properties towards selectivity of Man5GlcNAc2, thus generating a structure-specific detection tool. Using phage display to screen a diverse library of OAA […]
- by Mcnair, R., Whitfield, C. A., Poologasundarampillai, G., Jensen, O. E., Chernyavsky, I. L.Introduction: Stereological estimates of villous membrane thickness and surface area are widely used to infer the diffusive exchange capacity of the human placenta. A key geometric determinant of exchange capacity can be expressed as an effective diffusive length scale. Here we combine virtual histological sections with computational modelling in realistic villous geometries to assess the accuracy of classical stereological estimates of this diffusive length scale. Methods: Two terminal villi, reconstructed from three-dimensional imaging, were digitally sectioned to generate random two-dimensional […]
- by Pinto, A. J., Pradhan, B., Tetiker, D., Schmitt, M. P., Kim, E., Virnau, P.Loop extrusion by structural maintenance of chromosomes (SMC) complexes is essential for genome organization, yet the forces driving this process remain poorly understood. We present a coarse-grained model enabling predictive simulations of textit{in vitro} loop extrusion experiments at experimentally relevant time and length scales by matching parameters with concrete experiments. Using this model, we demonstrate that the extrusion forces generated by SMC motor proteins are just sufficient to overcome initial entropic barriers and sustain loop extrusion, highlighting that motors operate […]
- by Bairy, S. G., Prasad, T. K., Saravana Kumar, Y., Ganavi, B., S, S., S, S., Baskaran, S. P., Sounderrajan, V., Parthasarathy, K., Kamariah, N.The SARS-CoV-2 nucleocapsid (N) protein is central to genomic RNA recognition, condensation, and packaging, yet the molecular organization of its multivalent N-N and N-RNA interaction network involved in this process remains unclear. Here, we define the oligomerization and RNA-binding interfaces of the C-terminal domain (CTD) and its flanking intrinsically disordered regions (IDRs), the leucine-rich helix (LH) and the C-terminal IDR (C-IDR), using size-exclusion chromatography (SEC), cross-linking, mutational studies and NMR spectroscopy. We identify discrete oligomerization interfaces within the CTD and […]
- by Chin, A. C., Mukadum, F., Reynolds, M. J., Hocky, G. M., Alushin, G. M.Catch bonds, noncovalent supramolecular interactions whose lifetimes are increased by force, are ubiquitous in mechanical signaling pathways. The structural mechanisms of catch-bonding proteins remain unclear, hampering efforts to decipher how they are dysregulated in disease and exploit them therapeutically. The crosslinker -actinin-4 (ACTN4) forms catch bonds with actin filaments (F-actin) to support the function of kidney podocytes, and its force-insensitive K255E variant causes autosomal dominant focal segmental glomerulosclerosis (FSGS). Using cryo-electron microscopy (cryo-EM), we find that wild-type ACTN4 engages F-actin […]
- by Bouzos, N., Foley, S. L., Potamianos, A., Jacobs, C. O., Johnson, M. E., Zeno, W. F.The extent to which clathrin directly drives membrane curvature during endocytosis has remained a central, longstanding question in the field of endocytosis. Using a synthetic reconstitution system that recruits clathrin to lipid membranes independent of adaptor proteins, we demonstrate that clathrin alone can induce membrane fission and that its capacity to do so is governed by the mechanical properties of the lattice. Counterintuitively, conditions that strengthen lattice assembly suppress fission, whereas weakened assembly enhances fission despite reduced membrane association. Meso-scale […]
- by Jung, N., Park, H., Yang, J., Seok, C.Virtual screening has long been a central computational tool for rational ligand discovery, enabling the systematic prioritization of candidate molecules from large chemical libraries. Although docking and related approaches that explicitly account for receptor-ligand interactions have been developed and refined over several decades, achieving both reliable receptor-aware interaction modeling and computational scalability remains an open challenge, particularly for ultra-large chemical spaces. Ligand-based methods are fast and robust but do not explicitly incorporate receptor structure, whereas docking-based approaches model receptor-ligand interactions […]
- by Choudhuri, S., Adhikari, S., Mondal, J.Molecular dynamics (MD) simulations provide detailed insights into biomolecular motion but are often limited by the prohibitive cost of sampling long-timescale behavior. Here, we present a Transformer-based framework that reconstructs temporally continuous dynamical trajectories from only a small fraction of the initial data, directly targeting time-ordered evolution rather than independent ensemble snapshots. Using three systems spanning distinct dynamical regimes (intrinsically disordered -Synuclein, Cytochrome P450 ligand-binding motion, and a synthetic three-well potential), we show that the model learns both local fluctuations […]
- by Wang, S., Yourston, L., Maryu, G., Kim, Y., Walker, D., Kadiyala, U., Yang, Q.The temporal precision of biochemical oscillators is fundamentally constrained by the energy dissipated to suppress molecular fluctuations, a widely predicted trade-off governing information processing across biology and physics, from molecular motors to kinetic proofreading to computing. Yet, experimental validation in complex biological oscillators remains elusive due to challenges of systematically modulating energy while quantifying stochastic dynamics across large ensembles. Here, we establish a high-throughput droplet-microfluidics platform to reconstitute mitotic oscillations from Xenopus laevis egg extracts within thousands of sub-nanoliter compartments. […]
- by Fernandes, M., Huang, Y. X., Xu, I., Noguera Saigua, C., Li, J., Mahler, S.Cerebral blood volume (CBV) and blood flow (CBF) constitute key metrics for cerebrovascular monitoring, enabling assessment of stroke severity and risk-prediction, aging-related changes, and neurological diseases. CBF and CBV monitoring are key aspects in diagnosis, treatment triage, and clinical outcome of ischemic and hemorrhagic strokes. In recent years, there have been ongoing efforts toward the development of optical devices for noninvasive monitoring of CBV and CBF. Speckle contrast optical spectroscopy (SCOS) has recently emerged as a strong candidate for clinical […]
- by Azaldegui, C. A., Swasthi, H. M., Hu, L., Pulianmackal, L. T., Rivett-Trznadel, H., Liu, J., Vecchiarelli, A. G., Biteen, J. S.Carboxysomes are bacterial microcompartments that drive efficient carbon fixation in autotrophic bacteria. Critical to their function and inheritance is their spatial organization by the ParA-type ATPase, McdA, and its partner protein, McdB. Here, we investigate the -carboxysome McdAB system in Halothiobacillus neapolitanus using biochemical assays, quantitative fluorescence imaging, and mathematical modeling. We find that, unlike most ParA-type ATPases, the ATPase activity of McdA is only stimulated by DNA rather than by its partner protein McdB. Despite this difference, McdB conserves […]
- by Ramachandran, V., Potoyan, D. A.Protein-RNA phase separation gives rise to biomolecular condensates with rich internal organization, yet the molecular rules that connect sequence-encoded interactions and composition to the emergent architecture of these condensates remain poorly defined. Here, using large-scale residue-level coarse-grained simulations, we identify a molecular grammar that governs the formation and stability of multiphase protein-RNA condensates. We show that asymmetries in protein-protein and protein-RNA interactions, together with protein stoichiometry, chain length, and condensate density, collectively determine whether condensates adopt homogeneous, layered, biphasic, or […]
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