- by Skewes, B., McMahon, S., Auld, N. K., Marzano, N. R., van Oijen, A. M., Ecroyd, H.The Hsp70 chaperone system plays a central role in the folding of nascent polypeptides and in preventing protein misfolding and aggregation during cellular stress. However, the precise mechanism by which the human Hsp70, HspA8, remodels the conformations of individual chemically misfolded clients remains unclear. Moreover, it is unknown whether this mechanism differs from that used by Hsp70 to engage clients during heat stress to preserve native function. To address these questions, we report here the use of single-molecule fluorescence resonance […]
- by Cass, J. F., Wan, K. Y.For more than a billion years, microorganisms have evolved complex strategies for navigating aquatic habitats, despite the fundamental limitations and constraints imposed by their physical environment. A common theme across these strategies is the use of active slender appendages (cilia, flagella, archaella) to generate self-propulsion. Diverse selection pressures and evolutionary trajectories have driven the emergence of drastically different morphologies of biological microswimmers, each tailored for distinct functions ranging from motility to taxis to prey capture to feeding. Despite the biological […]
- by Biquet-Bisquert, A., Astezan, A., Marmol, M., Voyvodic, P. L., Mohite, N., Pedaci, F., Nord, A. L.Verapamil, a clinically used calcium channel blocker, enhances the activity of several tuberculosis antibiotics, but its mechanism of action and physiological effects on bacteria remain unresolved. A central debate concerns whether verapamil primarily inhibits efflux pumps or disrupts membrane energetics. Here, we use Escherichia coli as a model system to quantify single-cell and population-level physiological responses to verapamil with high temporal resolution. Real-time measurements of the rotational speed of individual flagellar motors, a single-cell proxy for the proton motive force […]
- by Wu, Q., Ciftci, D., Canul Tec, J., Reyes, N., Gregorio, G., Huang, Y., Boudker, O.Membrane transporters move polar solutes across lipid bilayers to regulate cellular metabolism, signaling, and drug distribution. These proteins operate via an alternating-access mechanism, cycling between extracellular-, intermediate-, and intracellular-facing conformations. The human excitatory amino acid transporter 1 (EAAT1) protects neurons from excitotoxic damage by mediating the uptake of glutamate and aspartate into glial cells. Defects in EAAT1 function result in numerous pathologies, including epilepsy and ataxia, suggesting that positive modulation of these transporters might ameliorate glutamate neurotoxicity. However, developing EAAT1 […]
- by Budhathoki, A., Pandey, G., Galeota-Sprung, J., Spille, J.-H.Single-molecule tracking measures the stochastic motion of individual biomolecules in the cellular environment. Statistical analysis of trajectory ensembles is required to gain insight into the biophysical nature of mobility states and molecular interactions that they reflect. Mobility states can be parameterized by a generalized diffusion coefficient and anomalous exponent. Experimental constraints such as finite track length and localization precision limit how accurately these parameters can be determined. We compare the performance of analysis methods to recover the input parameters from […]
- by Sukenik, N., Harris, C. C., Yadav, S., Chavez, M. S., Niman, C. M., Baczewski, L. T., El-Naggar, M. Y.Biological energy conversion relies on highly efficient electron transfer. The chirality induced spin selectivity (CISS) effect, which couples electron spin to momentum in chiral molecules, is hypothesized to promote this efficiency. While observed in isolated biomolecules, the physiological relevance of CISS during active cellular metabolism remains unknown. Here, we demonstrate that CISS influences extracellular electron transfer in living Geobacter sulfurreducens biofilms. Cultivation on ferromagnetic electrodes yields a significant asymmetry in respiratory current between opposite substrate spin states. Furthermore, in situ […]
- by Tyagi, N., Boodry, J., Chou, V., Snead, W. T., Shrinivas, K.Intrinsically disordered protein regions (IDPs) are found across the tree of life and characterized by the lack of a stable 3D fold, encoding function through a vast ensemble of conformations. This plasticity makes rational design of IDPs challenging. Physics-based approaches capturing distinct aspects of sequence composition, charge patterning, and molecular interactions have emerged as powerful predictors of ensemble-derived properties. Here, we present a machine learning framework for proteome-scale de novo IDP design by rationally inverting physics-based models. We first program […]
- by Zhang, H., Solis Fernandez, G., Louis, B., Vorsselmans, S., Hofkens, J., Kouwer, P. H. J., Yuan, H., Rocha, S.Cell migration in three-dimensional (3D) environments is highly plastic and regulated by extracellular matrix (ECM) cues. Engineered biomaterials provide controllable platforms to investigate how specific matrix signals regulate cell behavior in 3D, yet how defined biochemical signals control migration modes remain unclear. Here, we present tunable fibrous polyisocyanide (PIC) hydrogels functionalized with integrin-binding RGD peptides, cadherin-mimetic HAVDI peptides, or no ligands to direct mesenchymal, hybrid, or amoeboid-like migration of human adipose-derived stem cells without altering matrix mechanics. Using live-cell tracking, […]
- by Kenanoglu, C. U., Vardar, Y.Touchscreens have become the dominant interface in consumer electronics, yet interactions with them remain primarily visual. Incorporating haptic feedback that simulates touch sensations could make these interactions more natural and intuitive. Electrostatic actuation, which modulates friction by attracting the finger toward a capacitive surface using an alternating voltage, offers a promising approach. The resulting increase in friction is often attributed to the rise in real contact area; however, direct experimental evidence linking voltage input parameters to real contact area and […]
- by Naganathan, A. N., Madhan, H.The functioning of proteins is intimately linked to the conformational states they sample within the native ensemble. Generating ensembles from a single static structure is therefore a research domain receiving considerable attention. In this application note, we introduce Hashi, a pipeline to rapidly generate realistic structural ensembles from the outputs of the structure-based Wako-Saito-Munoz Eaton (WSME) statistical mechanical model of protein folding. This approach relies on integrating the block WSME model outputs – strings of zeros and ones describing the […]
- by Di Mambro, M., De Los Rios, P.Biomolecular condensates are thought to play a pivotal role in cellular organization by regulating biochemical reactants in space and time. Sustained molecular fluxes across condensate boundaries, together with the participation of phase-separating molecules in active chemical reactions such as ATP hydrolysis, call for a nonequilibrium description. Here, we propose a self-consistent framework in which diffusion–drift dynamics and chemical reactions are coupled through a conditional free energy, defined as the excess contribution to the chemical potential. Self-consistency is achieved by deriving […]
- by Lamb, J. R., Cardoso Mestre, M., Fenwyn Longrin, K., Bhat, P., Stevenson, M., Rhodes, A. D. Y., Gosieniecka, J., Redmond, L. C., Higgins, C. A., Rodriguez-Rodrigues, N., Lancaster, M. A., Manton, J. D.We present cleared-tissue direct-view oblique plane microscopy (CtDvOPM), which enables optically sectioned subcellular resolution imaging of centimetre-scale tissues at high-throughput over the full range of clearing media refractive indices (n = 1.33-1.56). CtDvOPM can image conventionally-mounted expanded, aqueous or non-aqueous cleared tissue samples at up to 2 m lateral by 14 m axial resolution over a 10 mm x 10 mm x 25 mm sample volume without image tiling, at up to 400 million voxels per second.
- by Hagarman, A., Franch, W. R., Oas, T. G.Surface factors that contribute to the virulence of Staphylococcus aureus have become therapeutic targets in the treatment of illness associated with this bacterium. Staphylococcal protein A (SpA) is a well-known contributor to S. aureus toxicity and virulence, although relatively little is known about protein A and how its biological function has evolved. SpA is displayed on the surface of the bacterium and contains 5 nearly identical helical ($approx{60}$ aa) domains that bind antibodies with high affinity ($K_dapprox{10}$ nM). The folding […]
- by Walter, N. G., Dai, L., Banerjee, P., Johnson-Buck, A., Blanchard, A., Li, Z.Background constrains analytical sensitivity: surveying larger sensor areas samples more analyte molecules but also accumulates false positives, limiting gains in detection performance. Here we introduce FINDER[-]Fluorogenic INstantaneous Digital Enumeration and Recognition[-]a single-molecule platform that combines kinetic fingerprinting with fluorogenic transient probes for rapid molecular classification under near-zero-background conditions. By suppressing both solution and surface-associated background at micromolar probe concentrations, FINDER classifies individual molecules within seconds-scale observation windows per field of view. This regime allows sensitivity to scale with surveyed sensor […]
- by Reese, S., de Ridder, W., van Hemmen, A., Mateescu, A.-G., Togo, R., Omi, S., Mavrakis, M., Richter, R., Koenderink, G. H.Septins are GTP-binding cytoskeletal proteins that shape and compartmentalize the plasma membrane. Their complex interactome has made it difficult to understand the molecular factors that govern their assembly. Moreover, it is unclear whether human septin hexamers and octamers form distinct higher-order assemblies, especially at the plasma membrane. Here, we address this question by using label-free methods to probe binding and self-assembly of purified human septins on supported lipid bilayers. Quartz crystal microbalance with dissipation (QCM-D) monitoring revealed that septin-membrane binding […]
- by Porcella, G., Sahin, A. T., Keller, J., Nawroth, J.Quantifying cilia-driven transport in mammalian tissues requires resolving rapid ciliary dynamics while preserving native three-dimensional geometry. Here we combine real-time four-dimensional light-field microscopy with tomographic particle tracking and physics-informed volumetric flow reconstruction to simultaneously image ciliary activity and reconstruct three-dimensional velocity fields in minimally dissected tissues. Applied across brain, respiratory, and reproductive epithelia, the workflow reveals recurrent vortical flow structures and enables quantitative analysis of flow-tissue coupling across individuals.
- by Garrels, C. S., Myers, J. B., Souza, S. A., Jarodsky, J. M., Reichow, S. L.Gap junction channels enable direct electrical and metabolic exchange between adjacent cells and tissues, where they organize into dense plaques containing tens to thousands of channels. Although plaque formation is known to modulate junctional conductance, the structural basis for channel-channel organization within a membrane environment remains poorly defined. Here, we reconstitute native lens connexin-46/50 (Cx46/50) gap junction channels into MSP-based lipid nanodiscs that incorporate multiple channels to create miniature plaque-like complexes suitable for single-particle cryo-electron microscopy (cryo-EM). We determine high-resolution […]
- by Ujma, J., Wheeldon, C., Schofield, A., Danby, M., Eatough, D., Bruton, D., Haris, A., Richardson, K., Langridge, D., Jarrell, A., Brown, J. M., Draper, B. E., Jarrold, M. F., Giles, K.Advances in Electrostatic Linear Ion Trap (ELIT) Charge Detection Mass Spectrometry (CDMS) over the past 10 years have revolutionized its use for analyzing very high-molecular-weight species such as protein complexes, viral vectors, vaccines, viruses, and amyloid fibrils. Nonetheless, ELIT-based CDMS has remained confined to a small number of specialized instrumentation groups, predominantly in academia, where large and complex home-built instruments are operated by highly skilled scientists in dedicated facilities. In this report, we discuss the primary challenges addressed in the […]
- by D'Asaro, D., Ciardo, D., Hyrien, O., Lacroix, L., Le tallec, B., Goldar, A., Audit, B., Arbona, J.-M.We present an analytical framework for modeling eukaryotic DNA replication that, given experimental Replication Fork Directionality (RFD) data, enables Bayesian inference of origin number, activation delay (t) and intrinsic timing ({lambda}), the mean replication time if each origin were isolated. By deriving closed-form expressions for RFD and Mean Replication Timing (MRT) under exponential and a specific Weibull firing-time distributions as functions of (t) and ({lambda}), we eliminate the need for stochastic simulations. These analytical results reveal that RFD, as a […]
- by Emenheiser, A. M., Gentry, E., Xue, H., Alvarez, P., O'Neill, K., Cao, K., Losert, W.The neurodegenerative disorder Alzheimer's disease (AD) is widely known for biomarkers such as amyloid beta plaques and tauopathy, as well as functional differences in memory and cognitive ability. Despite this devastating functional impact, a large body of work only focuses on molecular biomarkers of AD. In this study, we investigate collective neural dynamics in vitro and assess how network-level properties differ between a well-established model of familial AD (FAD) and a newly developed in vitro accelerated model (acAD). The new […]
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