- by Li, Z., Ren, Y., Kemkar, S., Mollenkopf, P., Kochanowski, J., Janmey, P. A., Purohit, P. K., Radhakrishnan, R., Vining, K. H.The mechanical properties of the extracellular matrix (ECM) regulate tumor growth and invasion in the tumor microenvironment. Models of biopolymer networks have been used to investigate the impact of elasticity and viscoelasticity of ECM on tumor behavior. Under tumor compression, these networks also show poroelastic behavior that is governed by the resistance to water flow through their pores. This work investigates the hypothesis that poroelastic properties regulate tumor growth. Here, alginate hydrogels with tunable ionic and hybrid ionic/covalent crosslinking are […]
- by Mandal, T., Roux, A., Garcia-Arcos, J. M.Flipper-TR is a membrane dye sensitive to lipid packing widely used to probe membrane tension in live cells via fluorescence lifetime imaging microscopy (FLIM). However, no consensus currently exists on the optimal strategy for extracting lifetime values, particularly across varying experimental setups and biological systems. Here, we systematically compare multiple approaches to estimate Flipper-TR lifetime, including multi-exponential reconvolution fitting, tail fitting, mean photon arrival time (first moment), and phasor analysis. These estimators are tested against changes in photon budget, sample […]
- by Punnoose, J. A., Kam, C. S., Melfi, T. J., Vangaveti, S., Chen, A. A., Halvorsen, K.Nucleic acid structures are stabilized by both base pairing and base stacking. While energetics of base pairing interactions are relatively well established, our understanding of the energetic contributions of base stacking remain incomplete. Here, we use a combination of single-molecule and computational biophysics approaches to investigate the effect of strand polarity on base-stacking energetics. We designed pairs of DNA constructs with reversed stacking polarities at nick sites, along with corresponding no-stack controls to isolate stacking contributions. Performing single-molecule force-clamp assays […]
- by Petalcorin, M. I. R.The mitochondrial proton motive force (PMF) underlies ATP synthesis, metabolite transport, and energy coupling. Yet, direct measurement of PMF remains technically challenging due to probe invasiveness, calibration drift, and compartmental averaging. Here, we introduce a physics-informed neural network (PINN) framework that reconstructs PMF from surrogate signals including NADH, oxygen, electron flux, proton leak, and reactive oxygen species (ROS). Using a synthetic curriculum dataset derived from biophysical ranges reported in the literature, our model achieved high predictive accuracy (R2; 0.99, RMSE […]
- by Johnson, J. A., Tosun, B., Yilmaz, M., Tolar, B., Yoshikuni, Y., Francis, C., Doukov, T., Yokoi, S., Wakatsuki, S., DeMirci, H.The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle in thaumarchaeota contributes significantly to global organic carbon fixation as the most energetically efficient aerobic carbon fixation pathway. The thaumarchaeal 3-Hydroxypropionyl-CoA Synthetase (ADP-forming; Nmar_1309) is crucial to this efficiency, utilizing ATP to ADP catalysis. This first reported structure of Nmar_1309 reveals a homodimer with a unique subdomain organization ([3-4-1-2-5]) and a distinct linker between subdomains 4 and 1. The presence of bound substrates including 3-hydroxypropionate, non-hydrolyzable ATP (ADPNP), and a phosphate suggests an intermediate state mimicking […]
- by Marszalek, P. E.DnaK is a prokaryotic Hsp70 chaperone with numerous functions in helping to fold nascent polypeptides and more generally in proteostasis. It also restores native structures to heat-shocked proteins in an ATP-hydrolysis-dependent manner. The structures of DnaK complexes with nucleotides, co-chaperones and small peptides have already been resolved. However, there are no structures of DnaK complexes with larger, mostly folded substrates, such as firefly luciferase (Fluc 61 kDa), which impedes the understanding of the mechanism through which DnaK refolds such large […]
- by Garcia, M., Reid, K. M., Robustelli, P.Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils that accumulate in pancreatic {beta}-cells of Type II Diabetes (T2D) patients. Recently discovered small molecules that modulate the kinetics of hIAPP amyloid formation could serve as starting points for developing T2D therapeutics, but no structural or mechanistic rationale exists to explain their binding mechanisms or effects on hIAPP aggregation pathways. Here, we utilize all-atom molecular dynamics computer simulations to elucidate the binding mechanisms of an hIAPP aggregation inhibitor (YX-I-1) and an aggregation […]
- by Bower, J. B., van der Velden, W. J. C., Gomez, K. P., Pan, M., Bumbak, F., Vaidehi, N., Ziarek, J. J.Detergents provide essential membrane mimetic environments for studying G protein coupled receptors (GPCRs), but their molecular impact on receptor energetics remains incompletely understood. We combined ligand binding, thermostability measurements and atomistic molecular dynamics to dissect detergent versus ligand driven stabilization in a thermostabilized neurotensin receptor 1 (enNTS1). Circular dichroism and ligand binding assays revealed that apo enNTS1 becomes progressively more stable in decyl maltoside (DM), dodecyl maltoside (DDM), and lauryl maltose neopentyl glycol (LMNG). Yet this gain in baseline stability […]
- by Yeltokov, A., Finegan, T. M., Fletcher, A. G., Lovegrove, H. E., Bergstralh, D. T.Oriented cell division is fundamental to development and tissue organization, requiring precise control of both spindle positioning and orientation. While cortical pulling forces mediated by dynein motor proteins are well-established drivers of spindle dynamics, the contribution of microtubule polymerization-based pushing forces remains unclear. We developed a generalizable computational biophysical model that integrates both pulling and pushing mechanisms to investigate spindle behavior across diverse cell types and geometries. This model successfully recapitulates experimental observations in three well-studied models: Drosophila follicular epithelial […]
- by Payne, A. M., Kaminow, B., MacDermott-Opeskin, H., Pulido, I., Scheen, J., Castellanos, M. A., Fearon, D., Chodera, J. D., Singh, S.Structure-based drug discovery technologies generally require the prediction of putative bound poses of protein:small molecule complexes to prioritize them for synthesis. The predicted structures are used for a variety of downstream tasks such as pose-scoring functions or as a starting point for binding free energy estimation. The accuracy of downstream models depends on how well predicted poses match experimentally-validated poses. Although the ideal input to these downstream tasks would be experimental structures, the time and cost required to collect new […]
- by Vasilenko, E. O., Ivankov, D. N.Many peptides and proteins self-assemble into large fibrillar aggregates, reaching sizes of several micrometers. This process typically involves nucleation, formation of transient oligomeric species ranging from dimers to assemblies comprising hundreds of monomers. The roles of these heterogeneous oligomers in initiating fibril growth vary significantly, as only a subset converts into primary nuclei, the smallest assemblies capable of spontaneous elongation. Consequently, the initial stages of peptide aggregation remain a critical challenge in elucidating amyloid fibril formation. Here, we analyze the […]
- by Rzaca, C., Kubisiak, A., Panek, D., Targosz-Korecka, M., Rajfur, Z.Substrate elasticity plays a pivotal role in regulating the morphology, mechanical properties, and cytoskeletal organization of cancer cells. In this study, we examined the response of U2OS osteosarcoma cells to substrates of varying stiffness, with a particular focus on cytoskeletal remodeling, cell elasticity, and microparticle internalization. To simulate environments of moderate and high stiffness, cells were cultured on polyacrylamide (PA) hydrogels with a stiffness of 40 kPa and on rigid glass substrates, respectively. Changes in cell morphology and cytoskeletal organization […]
- by Lira, R. B., Cavalcanti, R. M., Riske, K. A., Dimova, R.Membrane fusion is essential for signaling, cargo delivery, and synthetic cell growth, yet its mechanical consequences remain poorly defined. How fusion-driven membrane growth can be sustained without compromising compartment stability remains an unresolved challenge. Here, we established a minimal reconstituted system where content-loaded small liposomes fuse with single cell-sized giant unilamellar vesicles (GUVs), combining micropipette delivery, electrodeformation, and live imaging. Fusion outcomes were quantified through lipid and content mixing assays, GUV electrodeformation to track area and tension, and phase contrast […]
- by Madoo, K., Mazboudi, R., Kuschner, R., Gottlieb, P., Galarza, J. M., Khayat, R.Members of the Adenoviridae family routinely infect humans, exhibit significant genetic diversity, and are associated with a variety of illnesses. Types 4 and 7 frequently circulate in the United States and are major causes of respiratory disease. Infections can result in hospitalization and, in severe cases, death. Although a live wild-type-virus vaccine targeting these two types exists, its use is restricted to military personnel due to concerns about viral-shedding and the potential for genetic recombination. To overcome these limitations, we […]
- by Pott, E. D., Yang, M., Batey, J. E., Embree, J., Dong, B.Cell membranes display nanoscale heterogeneity in lipid composition and organization that regulates vital biological processes yet remains challenging to resolve with conventional imaging. We introduce spectral phasor single molecule localization microscopy (SP-SMLM), a hyperspectral and super-resolution method that combines wavefront-like optical filtering with single molecule imaging for simultaneous spatial and spectral analysis. A lab-built three-channel imager with sine/cosine filters encodes emission spectra of single molecules into the phasor space, enabling high-throughput, high-SNR mapping of membrane polarity at sub-50 nm spatial […]
- by Mueller, L., Coveney, S., Watson, I. L., Doring, A., Teh, I., Lwin, M., Dall'Armellina, E., Szczepankiewicz, F., Jones, D. K., Afzali, M., Schneider, J. E.PurposeCardiac diffusion weighted imaging (cDWI) commonly utilizes 2D-selective RF-pulses (such as ZOOMit) to avoid aliasing artifacts when reducing the field of view (FOV). These RF-pulses, which may require parallel transmit capability, typically take several 10s of milliseconds, prolonging the echo time (TE) of the imaging sequence. Conversely, slice selection using ZOnally-magnified Oblique Multi-slice (ZOOM) (i.e. tilting the excitation relative to the refocusing RF-pulse) is an alternative for reducing FOV while shortening TE. We hypothesized that ZOOM, with appropriate choices for […]
- by Kueppers, M., Moerner, W. E.Light microscopy remains indispensable in life sciences for visualizing cellular structures and dynamics in live specimens. Yet, conventional fluorescence imaging can suffer from phototoxicity, limited labeling efficiency, or perturbation of biological function. Label-free techniques such as interferometric scattering microscopy (iSCAT) offer a powerful alternative by detecting nanoscale structures based on their light scattering, without the need for dyes or genetic tags. iSCAT has enabled high-sensitivity detection of single proteins and viruses on clean surfaces. More recently, its application to live […]
- by Suprewicz, L., Byfield, F. J., Dutta, T. T., Janmey, P.Deformation of the nucleus often presents a barrier to cell migration through tight spaces, such as those encountered as cells move through tissues or across extracellular matrix barriers. Reorganization of the nucleus to allow its passage through spaces much smaller than its resting diameter requires forces generated by the cytoskeleton, as well as active reorganization within the nucleus driven by ATPases that crosslink or move chromatin. Here, we show that two different nuclear ATPases, the BRG1/SMARCA4 motor of the BAF […]
- by Dickwella Widanage, M. C., Perrone, B., Saha, J., Fu, R., Struppe, J., McGlinchey, R., Lee, J., Schurko, R., Ramamoorthy, A.Here, we report on the enhanced sensitivity of solid-state NMR experiments on -synuclein fibrils using a MAS cryoprobe. Nearly an order of magnitude improvement in signal-to-noise was observed in CPMAS, refocused-INEPT and 2D 13C-13C chemical shift correlation spectra of -synuclein fibrils acquired using a MAS cryoprobe compared to that of a conventional MAS probe.
- by Todisco, M., Jain, A.The stability of RNA base pairing and its limited four-letter code create an intrinsic potential for promiscuous RNA-RNA interactions. In vitro, such interactions drive RNA to self-assemble into aggregates. This raises a fundamental unanswered question: within a confined cellular volume at physiological mRNA abundances, how much aggregation would arise from sequence-encoded chemistry alone? Here, we establish this baseline with large-scale kinetic simulations of the E. coli transcriptome. Our simulations reveal that sequence-encoded base-pairing energetics is sufficient to generate a dynamic […]
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