- by Heymann, T., Oliinyk, D., Henneberg, L., Baggio Lorenz, M., Eikmeier, N., Thielert, M., Oeller, M., Grauvogel, L., Sitron, C. S., Loyd, B., Le Blanc, Y., Bloomfield, N., Batruch, I., Causon, J., Chelur, A., Ivosev, G., Tran, K., Talamantes, T., Schneider, B., Castro-Perez, J., Mann, M.Mass spectrometry-based proteomics increasingly demands platforms that combine quantitative rigor with the discovery capabilities of accurate mass systems. Here we present the ZenoTOF 8600 system, a compact mass spectrometry system that integrates enhanced ion capture and transmission optics with an optical detection system, Zeno trap-enhanced MS/MS, electron-activated dissociation, and scanning quadrupole data-independent acquisition (ZT Scan DIA). We show that ZT Scan DIA outperforms conventional variable-window DIA (Zeno SWATH DIA) in both identifications and quantitative reproducibility, and demonstrate the platform's versatility […]
- Contributions of error correction and the spindle assembly checkpoint to mitotic timing and fidelityby Ha, G., Qiu, L., Amir, A., Needleman, D.Chromosome segregation is a tightly-regulated process that normally occurs with high fidelity. Errors in chromosome segregation are associated with aging, cancer, and infertility. Initially erroneously attached chromosomes are corrected over the course of mitosis, with the spindle assembly checkpoint preventing entry into anaphase until this error correction is complete. Despite extensive work on the molecular basis of error correction and the spindle assembly checkpoint, it is still unclear how disruption of these processes contribute to chromosome segregation errors. Here, we […]
- by Kuba, S., Simpson, M. J., Buenzli, P. R.Biological tissues grow at rates that depend on the geometry of the supporting tissue substrate. In this study, we present a novel discrete mathematical model for simulating biological tissue growth in a range of geometries. The discrete model is deterministic and tracks the evolution of the tissue interface by representing it as a chain of individual cells that interact mechanically and simultaneously generate new tissue material. To describe the collective behaviour of cells, we derive a continuum limit description of […]
- by Vorobevskaia, E., Loerzing, P., Schlierf, M.Antibiotic multi-resistance (AMR) in bacteria poses a significant threat to global health, driven by mechanisms like the integron genetic system in Gram-negative bacteria. Integrons facilitate AMR by shuffling resistance genes through site-specific recombination, mediated by the integrase enzyme IntI. Earlier studies revealed that the mechanical stability of the synaptic complex, a structure formed by four integrase subunits and DNA, correlates with the recombination efficiency, and by extension, the adaptation capability. We identified a conserved C-terminal -helix in IntI that stabilizes […]
- by Desai, N. G., Garlapati, P., Borghese, C. M., Goldschen-Ohm, M. P.GABAA receptors (GABAARs) are pentameric ligand-gated ion channels (pLGICs) essential for inhibitory synaptic transmission throughout the central nervous system. Despite progress in understanding their three-dimensional structure, the molecular basis for how neurotransmitter binding is transduced to ion channel gating remains poorly understood. Furthermore, relatively little is known about the contributions of distinct subunits to this coupling within typical heteromeric receptors. A highly conserved proline (site 1) in the M2-M3 linker of pLGIC subunits is involved in channel gating — e.g., […]
- by Shiomi, S., Akiyama, K., Shiraiwa, H., Hamaguchi, S., Matsunaga, D., Kaneko, T., Hayashi, M.Developing active transport systems for microcargo delivery is challenging and requires overcoming the low Reynolds number constraints. We developed a bio-hybrid micro-swimmer, "chlamylipo" consisting of the green alga Chlamydomonas reinhardtii, encapsulated within a giant liposome. Although internal encapsulation offers cargo protection, it requires a mechanism to transmit the propulsion force across a closed membrane. We demonstrated that chlamylipo exhibited forward swimming and phototactic directional control. High-speed imaging of membrane shape and fluid flow revealed that the driving force originated from […]
- by Zakrzewska, S., Chen, Z., Park, E., Bhaskar, R. G., Bedell, T. A., Du Bois, J., Minor, D. L.Saxitoxin (STX) and its congeners (paralytic shellfish toxins, PSTs) are among the most potent small molecule toxins. PSTs are produced by harmful algal blooms and derive toxicity by disrupting voltage gated sodium channel (NaV) bioelectrical signaling. Understanding how PST structural variation affects target binding is crucial to develop means to counteract PSTs and exploit these natural products as drug development leads. Frog and toad saxiphilins (Sxphs) are soluble, high affinity STX toxin sponge proteins that offer a powerful platform to […]
- by Peters, J. B., Heidebrecht, C., Weber, M., Leutenegger, M., Hell, S. W.Investigating the movements and conformational changes of proteins in living cells is essential for understanding their function. The recently introduced fluorescence nanoscopy method called MINSTED has successfully tracked single fluorophore-labelled proteins, albeit in fixed cells and two dimensions only. Here we introduce a MINSTED setup for live-cell tracking of individual proteins in three dimensions (3D) with a localization precision {sigma} down to < 1 nm. Applied to the motor protein kinesin-1, our MINSTED nanoscope follows single proteins in 3D as […]
- by Gomez-Cruz, C., Gelin, M., Pradeau-Phelut, L., Munoz-Barrutia, A., Etienne-Manneville, S., Garcia-Gonzalez, D.Cells can experience time-varying mechanical cues, particularly when navigating through changing and complex microenvironments. Yet whether and how cells retain and use a short-term mechanical memory of recent deformations remains unclear. Here we show that, in glioblastoma cells, this memory is encoded by transient cytoskeletal anisotropy. Using uniaxial magneto-mechanical actuation aligned or perpendicular to the cell long axis, nanoindentation, and selective cytoskeletal perturbations, we find that distinct architectures of the actin cytoskeleton drive opposite mechanical responses: actin stress fibers mediate […]
- by McGrath, H., Cernekova, M., Kolar, M.The ribosomal exit tunnel, through which all nascent polypeptides emerge, is formed primarily by ribosomal RNA. Still, ribosomal proteins contribute roughly one quarter of the tunnel walls. In particular, proteins uL4 and uL22 define the narrowest region of the tunnel. This constriction, enriched in basic residues, mediates the earliest protein-protein contacts experienced by a nascent polypeptide. Here, we characterize its conformational dynamics by analyzing 222 Escherichia coli ribosome structures from the Protein Data Bank and by performing unbiased all-atom molecular […]
- by Blazquez, S., Yamauchi, M., Terakawa, T.DNA transcription is a complex process involving numerous components that can assemble into phase-separated transcriptional condensates. However, whether condensates formed by multiple transcription factors behave through simple additive effects or instead exhibit non-additive, emergent properties remains unclear. Here, we use large-scale molecular dynamics simulations to investigate how three core transcription factors regulating pluripotency and early embryonic development–Nanog, Oct4, and Sox2–organize biomolecular condensates in the absence and presence of DNA. We find that condensate formation is primarily driven by intrinsically-disordered-region-mediated interactions […]
- by Orlando, M. A., Shah, T., Faber, M. W., Bose, S., Orlando, B. J.The CbrA protein is a central regulator of carbon metabolism, biofilm formation, and virulence in Pseudomonas species, but the molecular mechanisms by which CbrA links nutrient sensing to downstream signaling has remained unclear. CbrA is a rare transceptor that combines membrane transporter and histidine kinase domains into a single functional polypeptide. The structural basis for histidine recognition and membrane transport, as well as signaling through intracellular histidine kinase domains has remained elusive. Here we determined a cryo-EM structure of CbrA […]
- by Gahlot, D., Castin, J., Mathur, S., Das, D., Kumar, A., Arun, A., Gain, C., Sharma, M., Pal, R. K., Jain, N., Biswal, B. K., Singh, R., Thukral, L.Membrane recruitment is a fundamental regulator of protein function. However, the allosteric mechanisms by which lipid binding controls protein activity remain poorly understood. In autophagy, the ubiquitin-like protein LC3 is lipid-anchored to autophagosomes, where it is essential for receptor recruitment and vesicle formation. While LC3-receptor interactions are structurally well defined, how membrane engagement governs LC3 functional dynamics has remained enigmatic. Here, we uncover that membrane binding triggers a major conformational transition in LC3, exposing functional pockets that are occluded in […]
- by Wang, X., Gan, J., Wu, W., Zhang, S., Zhang, T., Wang, C., Chen, Y., Zhang, Q., Wu, S.Osmotic pressure is a fundamental physical determinant of cellular homeostasis, and its perturbation is recognized as a critical factor in cancer progression and therapy. However, whether cancer cells and normal cells respond to osmotic pressure changes differently remains unclear. Here, we subjected cancer and normal cells to osmotic shocks and compared their volume and traction force changes. Under hypotonic conditions, cancer cells recovered their volume and forces much more slowly than normal cells, although both cell types responded similarly to […]
- by Rothfischer, F., Weiss, L., Wang, Y., Pauer, C., Lang, K., Yin, X., Amin, R., Lipfert, J., Liedl, T., Simmel, F. C., Tavacoli, J., Lak, A.Self-assembled DNA nanostructures show great promise as functional devices, highly configurable materials, and in nanorobotics. Magnetic control can provide a powerful actuation mechanism in a broad range of contexts, since it affords a high-level of external control, it is biocompatible, and orthogonal to chemical or electrical stimuli. Here we demonstrate magnetic molecular nanoactuators by leveraging the unique site-specificity of DNA origami to assemble highly anisotropic magnetic nanocubes on high-aspect ratio DNA origami bundles. We traced and controlled 100s of our […]
- by Rajasekaran, N., Toptygin, D., Liao, T.-W., Hilser, V. J., Ha, T., Kaiser, C. M.Molecular chaperones interact with non-native proteins, playing crucial roles in preventing misfolding and enable efficient folding in the cellular environment. Trigger factor is a bacterial chaperone that binds to ribosomes, interacting with nascent polypeptides emerging from the ribosome and guiding their early folding steps. In contrast to the central role of the chaperone in promoting folding of newly synthesized proteins, its dynamic interactions with nascent chains emerging from the ribosome remain poorly understood. Here, we use single-molecule fluorescence and optical […]
- by Udgaonkar, J., Kaushik, A.Protein unfolding invariably appears to be a cooperative transition; yet, the molecular basis by which structural elements could unfold in a coordinated manner remains unresolved. Here, the unfolding mechanism of the naturally occurring heterodimeric protein double-chain monellin (dcMN) was characterized using site-specific time-resolved FRET and fluorescence anisotropy decay measurements made under equilibrium conditions. Although ensemble-averaged measurements suggested an apparently cooperative transition, population-level analysis using the maximum entropy method coupled to time-resolved FRET revealed pronounced conformational heterogeneity, with partially contracted (N-like) […]
- by Desdorf, L. M., Stange, A. D., Damkier, H. H., Schioett, B., Praetorius, J., Duncan, A. L.The 3D structure and mechanism of action are unknown for the integral plasma membrane transport protein Solute Carrier 4A10, which has been characterized functionally as an electroneutral Na+:HCO3- cotransporter. We used structure prediction and molecular dynamics simulations to study the binding of the transported ions to the Solute Carrier 4A10 protein and suggest a model of sequential binding of Na+ followed by HCO3- to the ion binding domain. The binding of HCO3- to the protein appears to depend absolutely on […]
- by Morizet, J., Akemann, W., Mathieu, B., Leger, J.-F., Bourdieu, L.The ability to record 3D neuronal activity with cellular resolution, high signal-to-noise ratio (SNR) and millisecond temporal resolution is a major challenge in neuroscience. One powerful method is random-access two-photon microscopy based on acousto-optic deflectors (AODs), which uses a holographically-shaped point spread function (PSF) scanned in 3D to maximize the sampling rate and SNR. However, this approach suffers from greater background contamination due to the holographically shaped PSF than standard two-photon microscopy with diffraction-limited PSF. To overcome this limitation, we […]
- by Zhang, K., Peng, Z., Li, W., Wang, W.Biomolecular condensates play essential roles in cellular organization and are implicated in diverse pathological processes. Their formation is driven by liquid-liquid phase separation (LLPS), a process that requires coordinated multistep desolvation of biomolecular chains and multivalent inter-chain interactions. Although coarse-grained (CG) models with implicit solvent are widely used to probe LLPS thermodynamics and kinetics, they typically neglect explicit desolvation energetics, limiting their accuracy and mechanistic interpretability. Here, guided by all-atom simulations and experimental measurements, we develop a CG model that […]
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