- BioRxiv Biochemistry
- BioRxiv Bioinformatics
- BioRxiv Biophysics
- BioRxiv Cancer Biology
- BioRxiv Pharmacology and Toxicology
- BioRxiv Systems Biology
- BioRxiv Zoology
- 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 Lebek, N., Campellone, K.The decline of cellular proteostasis is a hallmark of aging and key contributor to neurodegenerative diseases. Protein turnover is controlled by the ubiquitin-proteasome and autophagosome-lysosome systems, but how degradation is coordinated when one of these pathways is compromised is not well understood. To study the regulation of proteostasis, we utilized human fibroblasts with targeted knockouts of the cytoskeletal factors WHAMM and JMY, which control multiple steps in autophagy. We found that cells lacking both WHAMM and JMY accumulated numerous intense […]
- by Delacruz, L. R., Ye, M., Libby, K. A., Han, K., Munger, C. J., Qiu, Y., Kurland, I. J., Ringel, A. E.Heterogeneity is a hallmark of biological systems, where cell-to-cell variability supports adaptation to changing environments, but also enables maladaptive states such as drug resistance. Many sources of non-genetic variation, particularly bioenergetics and metabolism, remain difficult to measure in living cells and connect to functional outcomes. Here, we introduce MARBL (Methionine Analogues for Ratiometric Bioenergetics in Live cells), a method that encodes translationally-coupled energetic responses to metabolic stress as an internally normalized signal within the surface proteome of living cells. Applying […]
- by Siebeneichler, B., Liu, X., Rodriguez Cruz, P. E., Naser, D., DelMistro, G., Steckner, J., Schaefer, A., Tran, N., Holyoak, T., Meiering, E. M.Protein aggregation is of broad importance in biotechnology and disease, yet the structural heterogeneity of cellular aggregates has confounded high-resolution structural analysis. Inclusion bodies (IBs) formed in Escherichia coli are an attractive, controllable system for unravelling the complexities of protein aggregation in a cellular context. Here, a multimodal analysis integrating residue-resolved quenched amide hydrogen-deuterium exchange (qHDX), proteolysis, FTIR, Congo red binding, and chemical denaturation is applied to IBs formed by proteins encompassing stable beta- and alpha- globular folds, a partially […]
- by Jalkut, S. L., Bischoff, L. M., Pitsch, M. J., Losick, V. P.In the animal kingdom, copulatory wounds occur due to aggressive behavior or injury from the female or male appendages in contact. Copulatory wounds are a source of sexual conflict as the injury can lead to death or infection. In the laboratory, Drosophila melanogaster does not ordinarily exhibit signs of injury during copulation, but here we find can be induced to do so by overexpression of the serine protease, Hayan, in either the male or female fruit fly. Melanin deposits mark […]
- 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 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 Gallego-Murillo, J. S., van Lakwijk, I., Yagci, N., Reisz, J. A., Pozo Garcia, V., D'Alessandro, A., van der Wielen, L. A. M., von Lindern, M., Wahl, S. A., Van den akker, E.Transfusion-ready red blood cells (RBC) can be cultured ex vivo from hematopoietic progenitors. Despite its promising outlook, a cultured RBC transfusion unit cannot be produced at competitive costs. Large volumes of medium are required to maintain a maximum erythroblast cell density of 1-2.106 cells/mL during the proliferation stage of the culture. To identify the origin of the cell density limitation, we compared the growth support and changes in the cellular metabolomic signature while using different media formulations and feeding regimens. […]
- by Pajonk, O., Albert, L., Schafer, J. A., de Jager, L., Martin de Hijas, C., Papagiannidis, D., Odehnalova, K., Friemel, N., Esch, B. M., Frohlich, F., Luzarowski, M., Borner, G., Forster, F., Schuck, S.ESCRT proteins remodel membranes at many cell organelles, including the endoplasmic reticulum (ER). Here, we investigate whether ESCRTs in budding yeast participate in stress-induced ER reorganisation. We find that ER stress triggers the formation of tubular ER subdomains that recruit various ESCRT proteins. Recruitment of the major ESCRT-III protein Snf7 is mediated by the ESCRT-associated protein Bro1, a homologue of human ALIX, in a manner that is mechanistically distinct from Bro1 function at endosomes. ESCRT-containing ER subdomains are derived from […]
- by Cong, Y., Zhou, X., Shen, L., Yuan, X., Chen, C., Wang, T., Li, Z., Peng, J., Lang, X., Ye, X., Chen, T., Chen, K., Su, C., Huang, Z.Mature mammalian sperm rely on active proteasomes containing the testis-specific subunit 4s for capacitation, acrosomal exocytosis and fertilization, but how these proteasomes are structurally specialized remains unclear. We determine the endogenous proteasome landscape of mature bovine spermatozoa and testis using cryo-electron microscopy. We resolve five sperm proteasome assemblies: PA200-20S, free 20S, a previously unrecognized PA200-20S-PAC1/2 hybrid, and PAC1/2-20S complexes capped at one or both ends. Unexpectedly, the PAC1/2-bound complexes are mature and POMP-free, selectively incorporated for 4s, lack 2, and […]
- by Hua, J., Krystofiak, E. S., Pumford, A. D., Page-McCaw, A., Hutson, M. S.Tissue wounds comprise both dead and damaged cells. In epithelial wounds, repair is accomplished by cells at the wound edges, which are themselves often damaged. In the Drosophila pupal notum, wound-adjacent epithelial cells with plasma membrane damage often fuse to form syncytia; when plasma membrane damage is prevented, syncytia do not form. Damaged cells share cytoplasm as soon as milliseconds after wounding, and fusion pores connecting cell membranes form minutes later. A genetic screen reveals that wound-induced fusion requires endocytosis […]
- by Araten, A. H., Ho, E. K., Toettcher, J. E.In canonical developmental patterning, the embryo is exposed to gradients of signaling activators that elicit different cellular responses depending on the activator's concentration. Recent optogenetic studies of terminal ERK signaling downstream of Torso receptor tyrosine kinase in the early Drosophila embryo reveal that even a brief, 5-minute ERK stimulus is sufficient to rescue the development of larval "tail" structures. Here, we reveal components of the molecular network that defines this sensitive developmental fate response. We find that low ERK doses […]
- by Hwang, I.-J., Kim, J., Patel, A., Zhang, L., Miller, J., Piletsky, S., Clift, C. L., Hisey, C. L., Kim, Y., Kim, M.Extracellular vesicles (EVs) carry molecular signatures of their originating cells and have thus emerged as promising biomarkers. However, their clinical utility remains limited due to their low abundance and the modest sensitivity of current EV detection methods in complex biological environments. Here, we present a quantum well defect functionalized carbon nanotube sensor coupled with integrin-recognition RGD tripeptide for EV detection in human plasma. Leveraging the abundance of integrins on EV surfaces, we targeted a5b1, aVb1, and aVb3 subtypes. The nanosensor […]
- 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 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 Huang, H.-W., Tyrlik, M., Huang, P.-W., Yeo, A. I., Kim, A.-R., Perrimon, N., Tseng, C.-Y., Bellen, H. J., Ryoo, H. D.AP-2 is a key mediator of clathrin-mediated endocytosis (CME) that internalizes cargo from the plasma membrane. NECAP proteins physically bind phosphorylated AP-2 during CME, but their roles during endocytosis remain unresolved, with conflicting reports about their function. Here, we report that Drosophila NECAP is dispensable for development, but antagonizes light-dependent Rhodopsin-1 (Rh1) internalization, a process that occurs through AP-2-mediated endocytosis. Specifically, loss of Drosophila NECAP causes excessive light-dependent Rh1 internalization and an age-related retinal degeneration that can be rescued by […]
- 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 Roth-Carter, R., Helms, E., Saldivar, J. C., Podrabsky, J.Hypoxia and anoxia are known to suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus show a strong tolerance to extended anoxic exposure, indicating an improved genomic stability under oxygen starvation. Here we investigate the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxic exposure. Live cell imaging confirms continued cell proliferation of WS40NE cells for the first 24 hours of […]
- 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 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.