- by Rout-Pitt, N., Barnes, S., Reyne, N., McCarron, A., Donnelley, M., Kostecki, R., Noschka, E.We have developed a novel biosensing device that can detect the real-time, dynamic state of in vivo oxidative status (IVOS) in living systems. Oxidative stress is a well-established condition in CF animal models and humans. Elevated oxidative stress conditions are associated with excessive inflammatory responses from neutrophils that result in fibrotic tissue formation. As such, numerous clinical and preclinical studies suggest that elevated oxidative stress, combined with the heightened pro-inflammatory milieu observed in CF phenotypes, likely increases susceptibility to recurrent […]
- A Comparison of Computational Methods for Modeling Stochastic Collaborative DNA Methylation Dynamicsby Lemus, M. A. G., Read, E. L.The methylation of cytosine-phosphate-guanine (CpG) dinucleotides in DNA is linked to gene expression, cancer, and aging. The methylation levels of nearby CpG sites influence many enzymatic reactions governing DNA methylation. However, efficiently modeling this influence, known as "collaborative" DNA methylation, is computationally challenging. We compare the efficiency and accuracy of three collaborative DNA methylation modeling approaches: a stochastic simulation algorithm (SSA), an exact Chemical Master Equation (CME), and a mean-field CME. The exact CME model provides high accuracy but is […]
- by Imamura, R., Nakane, Y., Kataoka, N., Abe, H., Ohshima, T., Sotoma, S., Sugi, T.Subtle deviations in biophysical parameters, particularly temperature, within deep-brain regions exert substantial effects on whole-body homeostasis and metabolism. However, the underlying mechanisms remain poorly understood because cellular-resolution measurements of these parameters in the deep brain have been technically inaccessible. Here, we present a lensless fiber-optic quantum-sensing endomicroscopy that enables cellular-resolution temperature mapping in the mouse deep brain. A microwire-coupled, lensless fiber bundle permits simultaneous temperature readout from optically detected magnetic resonance spectra of >50 fluorescent nanodiamonds, achieving cellular resolution (4.88 […]
- by Lionnet, T., Clark, F. T., Whitney, P. H., Saiz, N., Ziarno, A.Single-molecule localization microscopy (SMLM) captures nanoscale detail with fluorescence imaging. As large-area cameras and multiplex imaging become standard, chromatic errors are growing more complex, challenging precise analysis. To address this challenge, we present BeadBuddy, an open-source, user-friendly software that uses images of fluorescent beads to model and correct 3D, spatially varying chromatic errors. BeadBuddy achieves sub-voxel resolution in DNA Fluorescence In Situ Hybridization (FISH) and is applicable across SMLM modalities.
- by Rudnizky, S., Murray, P. J., Sorensen, E. W., Koenig, T. J. R., Pangeni, S., Merino-Urteaga, R., Chhabra, H., Caccianini, L., Davidson, I. F., Osorio-Valeriano, M., Hook, P. W., Meneses, P., Hao, J., Zarb, J. S., Hatzakis, N. S., Timp, W., Farnung, L., Vos, S. M., Peters, J.-M., Aksimentiev, A., Ha, T.CCCTC-binding transcription factor (CTCF) and the cohesin complex shape the genome into loops and topologically associating domains (TADs), yet the mechanisms linking CTCF dynamic behavior to its function as a cohesin barrier remain unclear. Using integrated experimental and computational approaches, we demonstrate that individual CTCF-DNA complexes are intrinsically mobile over distances and timescales compatible with cohesin capture during loop extrusion, driven by the dynamic rearrangements of zinc finger (ZF) domains. Genome-wide single-molecule accessibility and sequencing analyses further reveal that these […]
- by Miotto, M., Bo', L., Ruocco, G., Di Angelantonio, S., Basilico, B.SYNGAP1 mutations have been linked to a range of neuropathological disorders and, more recently, to the insurgence of cancer. Despite its emerging relevance, the molecular basis of SynGAP's action remains poorly understood. Here, using extensive molecular dynamics simulations integrated with structural analysis, we define how the SynGAP C2 domain associates with lipid bilayers. In particular, we observed spontaneous membrane binding of the domain in two distinct orientations, top and side, defined by the relative positioning of the C2 domain to […]
- by Subirachs, V. V., Syevda, T., Maltsev, A. V., Yang, D., Lakatta, E. G., Stern, M. D., Maltsev, V. A.The sinoatrial node is the primary cardiac pacemaker. Individual sinoatrial node cells (SANCs) generate spontaneous rhythmic action potentials (APs) that initiate each heartbeat. The mechanism of SANC automaticity and its modulation by autonomous nervous system are based on the coupled function of molecules of both the cell membrane (ion channels, exchangers, and pumps) and the sarcoplasmic reticulum (SR), which generates rhythmic local Ca releases (LCRs). While LCRs are generated by ryanodine receptors (RyRs), the molecular-scale structure of the RyR network […]
- by Otwinowski, Z., Bromberg, R., Guo, Y., Borek, D.Current guidelines for depositing cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) data require submission of unfiltered, unmasked, and unsharpened raw half-maps. The Fourier Shell Correlation (FSC) between the half-maps is then used as a proxy for the signal-to-noise ratio (SNR) to estimate the reconstruction's resolution. This policy was introduced to enable independent validation of reported resolutions. Although developed to safeguard data integrity and minimize bias, these guidelines do not account for specific features of modern cryo-EM processing software, in […]
- by Brouillon, C., Pushkarev, A., Lienard, M. A., Hwang, S., Munhoven, A., McDowell, R. J., Lucas, R. J. J., Porter, M., Sun, H., Hegemann, P.Of all visual systems shaped over billions of years, stomatopod crustaceans or mantis shrimps possess arguably one of the most extraordinary eyes, with unmatched potential for broad colour detection (T. W. Cronin & Marshall, 1989; J. Marshall & Oberwinkler, 1999; J. Marshall et al., 2007). Yet how their unusual diversity of visual opsins (Porter et al., 2020) maps onto functional spectral sensitivities (Streets et al., 2022; T. W. Cronin et al., 2022) has remained unresolved. Here, we uncover the molecular […]
- by Sen, A., Nakamura, T., Terashi, G., Kim, K., Bhatt, V., Chellam, S., Tran, L., Kihara, D.The highly regulated assembly of different proteins forming mature P1 myophage capsid show a unique polymorphism where the virion population is primarily divided in brackets of two different sizes. The contrasting capsids, sized 95nm (capsid_L), triangulation number (T)=13 and 68nm (capsid_S) with T=7, both in Dextro format and have the same protein forming the phage head with similar intra and inter capsomeric interactions. Comparative study of the electron density maps of the capsids reveals the presence of protein appendages DarA […]
- by Ge, Z., Ni, Q., Yan, Y., Wu, Y., Si, B. R., Fu, J., LIN, D., Konstantopoulos, K., Li, Y., Sun, S.The membrane potential of eukaryotic cells, classically described by the Goldman-Hodgkin-Katz model, is conventionally attributed to the steady-state balance of transmembrane ionic fluxes. Here, we demonstrate that non-excitable cells maintain stable, spatially heterogeneous membrane voltage gradients. Using the ratiometric voltage indicator JEDI-2P-cyOFP1, we quantitatively map membrane potential in live cells and find that cellular protrusions are consistently depolarized, whereas lateral membrane regions are hyperpolarized. These voltage gradients strongly correlate with anisotropic distributions of anionic lipids, including phosphatidylserine (PS) and phosphatidylinositol […]
- by Mu, J., Lai, L.Intrinsically disordered proteins (IDPs) play crucial roles in biomolecular condensate formation. While molecular dynamics simulations employing coarse-grained models have emerged as useful tools for studying IDP phase behavior, current force-field parameterizations remain limited in their ability to simulate post-translational modifications (PTMs), which are the critical regulatory elements of IDP involved condensation with profound biological implications. To address this gap, we developed interaction parameters for five common PTM types: phosphorylated serine (pSer), threonine (pThr), and tyrosine (pTyr); acetylated lysine (AcLys); and […]
- by Dillon, S., Armiger, A., Murgoci, A., Skingle, L., Tabegna, F. G. A., McDonald, S., Mumm, S., Whyte, M. P., Garton, M., Poole, K. E. S., Duer, M. J.Bone mineral molecular architecture is tightly regulated by the kinetics of calcium phosphate phase transformations. In the rare skeletal disease hypophosphatasia (HPP), caused by inactivating mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNSALP), accumulation of inorganic pyrophosphate (PPi) alters these phase dynamics. Using solid-state nuclear magnetic resonance spectroscopy and high-resolution electron microscopy, in patient samples we show that bone mineral from compound heterozygous HPP patients exhibits a loss of hydrated amorphous interfacial phases and instead contains highly crystalline […]
- by Tanaka, K., Nishikawa, K., Shiimura, Y., Fujiyoshi, Y., Tsutsumi, N.CC chemokine receptor 7 (CCR7), which orchestrates adaptive immunity, exhibits a phenomenon known as biased agonism. CCL19 induces robust G protein signaling and {beta}-arrestin recruitment, leading to transient signaling. In contrast, CCL21 preferentially activates G protein pathways with minimal arrestin engagement, resulting in sustained signaling and differential functional outcomes. Here, we present the cryo-EM structures of the human CCR7-Gi complex with either CCL19 or CCL21. The structures reveal that while both engage a conserved orthosteric pocket, they adopt markedly distinct […]
- by Seo, L., Farran, I., Aslam, A., Li, X., Jaishankar, P., Fraser, J. S., Renslo, A., Wankowicz, S. A.Structure-based drug design has traditionally focused on optimizing static, enthalpic interactions between ligands and proteins or on displacing binding site solvent molecules to entropically favor binding. A potentially large contributor to binding thermodynamics is the difference in conformational entropy of the protein upon binding a ligand; however, this information has been difficult to quantify especially in high throughput. Here, through multiconformer ensemble modeling of hundreds of ligand-bound SARS-CoV-2 Macrodomain (Mac1) X-ray structures, we show how ligand binding reorganizes both protein […]
- by Dec, R., Dzwolak, W., Winter, R.Adenosine triphosphate (ATP), a common constituent of protein-rich biomolecular condensates, itself undergoes liquid-liquid phase separation (LLPS) even in the absence of polypeptides. Using polyethylene glycol as macromolecular crowding agent, we observed robust ATP droplet formation at pH 2-11. Moderate NaCl and lower temperatures promote LLPS by lowering critical ATP and crowder concentrations. Very high salt reverses this trend through anomalous underscreening, but ATP droplets still survive in hypersaline environments. Most importantly, physiological millimolar ATP concentrations are sufficient for phase separation […]
- by Imtiaz, Y., Hardin, J., Sheyitov, B., Zhang, L., Foote, A., Rahman, M. H. b. A., Jackson, K., Piranej, S., Bazrafshan, A., Salaita, K.Nucleic acid-based synthetic motors emulate key behaviors of biological machines, enabling applications in biosensing and nanoscale actuation. Among the reported synthetic motors, RNase H-powered motors offer high speed and processivity with demonstrated applications in computation and viral sensing. However, these motors rely on RNA as "fuel" source, limiting their stability. Here, we report the development of a robust, RNA-free DNA motor powered by Exonuclease III. These motors exhibit self-avoiding rolling motion driven by enzymatic hydrolysis of surface-bound DNA fuel strands, […]
- by Agarwal, T., Sneideris, T., Svara, F., Jermakovs, K., Coyle, H., Qamar, S., Kava, E., Scrutton, R., Pleschka, N., Peres, P., Cereghetti, G., Andrzejewska, E., Diaz-Barreiro, A., Palmer, G., Costa-Filho, A. J., Krainer, G., Knowles, T. P., Nixon-Abell, J.Biomolecular condensates are membraneless compartments, crucial for organising and regulating diverse cellular processes. Current approaches to study condensate biology either use simplified recombinant protein systems with limited physiological relevance, or complex live-cell models with restricted experimental control and scalability. Here, we present ExVivo PhaseScan, a droplet microfluidics platform that couples mammalian lysate-based reconstitution with scalable analysis to generate high-resolution phase diagrams of physiological protein condensates. We apply this approach to study two complex multicomponent condensate systems, stress granules and nucleoli, […]
- by V, V. K., Garai, K.Aberrant liquid to solid transition (LST) of tau is a pathological hallmark of several tauopathies, most notably Alzheimers disease (AD). ApoE4 is a major genetic risk factor for AD and has been shown to exacerbatetau pathology, although the underlying mechanismremains unclear. Herewe investigate whether apoE isoforms can influenceamyloid aggregation of tau occurring via the liquid-liquid phase separation (LLPS) pathway by monitoring individual tau condensates in real time using total internal reflection fluorescence microscopy (TIRFM) and confocal microscopy. We find that […]
- by Do, H. N., Shanker, A., Kidner, R. Q., Montoya, M. M., Kubicek-Sutherland, J. Z., Gnanakaran, S.Signal peptides (SPs) are short amino acid sequences found at the N-terminus of nascent polypeptides, which serve critical roles in trafficking, folding, and post-translational processing of mature proteins. Many sequence-based computational methods have been developed to predict and design optimal SPs for protein secretion in human cells. In this work, we introduce a structure-based workflow for identifying SPs and their regions, aiming to optimize SPs for protein secretion in human cells. Structural modeling of the SPs in complex with human […]
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