Score: 42.9, Published: 2024-02-12
DOI: 10.1101/2024.02.12.579607
Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, for particles smaller than 50kDa, single particle cryo-EM often fails due to the limited imaging contrast of individual micrographs. For dose-resilient samples often studied in the physical sciences, electron ptychography - a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) - has recently demonstrated resolution down to tens of picometers for thin specimens imaged at room temperature. Here we applied ptychographic data analysis to frozen hydrated single protein particles, reaching sub-nanometer resolution 3D reconstructions. We employed low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high speed of the electron detector allowed us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan position, from which the scattering potentials of the samples were iteratively reconstructed. The reconstructed micrographs show strong contrast enabling the reconstruction of the structure of apoferritin protein at up to 5.8 [A] resolution. Ptychography is a promising tool to study the structure of smaller frozen hydrated protein particles, and, once combined with electron tomography tilt series, bears the potential to provide unique insight into the ultrastructure of vitrified biological tissue.
Score: 62.2, Published: 2024-02-11
DOI: 10.1101/2024.02.06.579238
Sample loss due to air-water interface (AWI) interactions is a significant challenge during cryo-electron microscopy (cryo-EM) sample grid plunge freezing. We report that small Late Embryogenesis Abundant (LEA) proteins, which naturally bind to AWI, can protect samples from AWI damage during plunge freezing. This protection is demonstrated with two LEA proteins from nematodes and tardigrades, which rescued the cryo-EM structural determination outcome of two fragile multisubunit protein complexes.
Score: 23.2, Published: 2024-02-11
DOI: 10.1101/2024.02.09.579083
navigate is a turnkey, open-source software solution designed to enhance light-sheet fluorescence microscopy (LSFM) by integrating smart microscopy techniques into a user-friendly framework. It enables automated, intelligent imaging with a Python-based control system that supports GUI-reconfigurable acquisition routines and the integration of diverse hardware sets. As a comprehensive package, navigate democratizes access to advanced LSFM capabilities, facilitating the development and implementation of smart microscopy workflows without requiring deep programming knowledge or specialized expertise in light-sheet microscopy.
Score: 20.4, Published: 2024-02-11
DOI: 10.1101/2024.01.03.574123
Fascin crosslinks actin filaments (F-actin) into bundles that support tubular membrane protrusions including filopodia and stereocilia. Fascin dysregulation drives aberrant cell migration during metastasis, and fascin inhibitors are under development as cancer therapeutics. Here, we use cryo-electron microscopy, cryo-electron tomography coupled with custom denoising, and computational modeling to probe fascins F-actin crosslinking mechanisms across spatial scales. Our fascin crossbridge structure reveals an asymmetric F-actin binding conformation that is allosterically blocked by the inhibitor G2. Reconstructions of seven-filament hexagonal bundle elements, variability analysis, and simulations show how structural plasticity enables fascin to bridge varied inter-filament orientations, accommodating mismatches between F-actins helical symmetry and bundle hexagonal packing. Tomography of many-filament bundles and modeling uncovers geometric rules underlying emergent fascin binding patterns, as well as the accumulation of unfavorable crosslinks that limit bundle size. Collectively, this work shows how fascin harnesses fine-tuned nanoscale structural dynamics to build and regulate micron-scale F-actin bundles.
Score: 12.9, Published: 2024-02-12
DOI: 10.1101/2024.02.12.579899
Biomolecular condensation via phase separation of proteins and nucleic acids has emerged as a crucial mechanism underlying the spatiotemporal organization of cellular components into functional membraneless organelles. However, aberrant maturation of these dynamic, liquid-like assemblies into irreversible gel-like or solid-like aggregates is associated with a wide range of fatal neurodegenerative diseases. New tools are essential to dissect the changes in the internal material properties of these biomolecular condensates that are often modulated by a wide range of factors involving the sequence composition, truncations, mutations, post-translational modifications, and the stoichiometry of nucleic acids and other biomolecules. Here, we employ homo-Forster Resonance Energy Transfer (homoFRET) as a proximity ruler to study intermolecular energy migration that illuminates the molecular packing in the nanometric length-scale within biomolecular condensates. We used the homoFRET efficiency, measured by a loss in the fluorescence anisotropy due to rapid depolarization, as a readout of the molecular packing giving rise to material properties of biomolecular condensates. Using single-droplet anisotropy imaging, we recorded spatially-resolved homoFRET efficiencies of condensates formed by fluorescent protein-tagged Fused in Sarcoma (FUS). By performing single-droplet picosecond time-resolved anisotropy measurements, we were able to discern various energy migration events within the dense network of polypeptide chains in FUS condensates. Our homoFRET studies also captured the modulation of material properties by RNA, ATP, and post-translational modification. Additionally, we utilized mammalian cell lines stably expressing FUS to study nuclear FUS and oxidative stress-induced stress granule formation in the cytoplasm. Our studies demonstrate that spatially-resolved homoFRET methodology offers a potent tool for studying intracellular phase transitions in cell physiology and disease.
Score: 10.4, Published: 2024-02-13
DOI: 10.1101/2024.02.12.579963
The widespread adoption of cryoEM technologies for structural biology has pushed the discipline to new frontiers. A significant worldwide effort has refined the Single Particle Analysis (SPA) workflow into a reasonably standardised procedure. Significant investment of development time have been made particularly in sample preparation, microscope data collection efficiency, pipeline analyses and data archiving. The widespread adoption of specific commercial microscopes, software for controlling them and best practises developed at national facilities has also begun to establish a degree of standardisation to data structures coming from the SPA workflow. There is opportunity to capitalise on this moment in the fields maturation, to capture metadata from SPA experiments and correlate this with experimental outcomes, which is presented here in a set of programmes called EMinsight. This tool aims to prototype the framework and types of analyses that could lead to new insights into optimal microscope configurations as well as for defining methods for metadata capture to assist with archiving of cryoEM SPA data. We also envisage this tool to be useful to microscope operators and facilities looking to rapidly generate reports on SPA data collection and screening sessions. SynopsisEMinsight is a Python-based tool for systematically mining metadata from single particle analysis cryoEM experiments. The capture and analysis of metadata facilitates assessment of instrument performance, provides concise reporting of experiment performance and sample quality by analysing preprocessing results, and gathers metadata for deposition. We envisage this approach to benefit the microscope operator, facility managers, database developers and users.
Score: 6.4, Published: 2024-02-13
DOI: 10.1101/2024.02.12.579954
The basic zippers (bZIPs) are one of two large eukaryotic families of transcription factors whose DNA binding domains are disordered in isolation but fold into stable -helices upon target DNA binding. Here we systematically disrupt pre-existing helical propensity within the DNA binding region of the homodimeric bZIP domain of cAMP-response element binding protein (CREB) using Ala-Gly scanning and examine the impact on target binding kinetics. We find that the secondary structure of the transition state strongly resembles that of the unbound state. The closest residue to the dimerisation domain that has been examined is largely folded within both unbound and transition states; dimerisation apparently propagates additional helical propensity into the basic region. The results are consistent with electrostatically-enhanced DNA binding, followed by rapid folding from the folded zipper outwards. Interestingly, despite taking the exact experimental approach suggested for testing it, we find no evidence for disorder-mediated rate enhancement predicted by fly-casting theory.
Score: 6.5, Published: 2024-02-09
DOI: 10.1101/2024.02.07.579285
Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1) with DNA contributes to the formation of condensed chromatin states. HP1 localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1 and dictating emergent functions of its condensates, remains only partially understood. Here, we leverage a reductionist system, comprised of modified and unmodified histone H3 peptides, HP1 and DNA to examine the contribution of specific epigenetic marks to phase behavior of HP1. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1 condensates, while peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1 phase separation. In addition, inspired by the decreased ratio of nuclear H3K9me3 to HP1 detected in cells exposed to uniaxial strain, using fluorescence microscopy and rheological approaches we demonstrate that H3K9me3 histone peptides modulate the dynamics and network properties of HP1 condensates in a concentration dependent manner. These data suggest that HP1-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells in response to mechanostimulation. Statement of significanceThe organization of genomic information in eukaryotic cells necessitates compartmentalization into functional domains allowing for the expression of cell identity-specific genes, while repressing genes related to alternative fates. Heterochromatin hosts these transcriptionally silent regions of the genome - which ensure the stability of cell identity -and is characterized by repressive histone marks (H3K9m3) and other specialized proteins (HP1a), recently shown to phase separate with DNA. We show that HP1a forms condensates with DNA which persist in the presence of H3K9me3 peptides. The viscoelastic nature of these condensates depend on H3K9me3:HP1 ratios, which are modulated by mechanical strain in cells. Thus, phase separation may explain the dynamic behavior of heterochromatin in cells, in response to mechanostimulation.
Score: 6.0, Published: 2024-02-12
DOI: 10.1101/2024.02.09.579714
Myosin-Is colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by Arp2/3 complex on the surface of beads coated with myosin-I and the WCA domain of N-WASP. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Remarkably, myosin-I triggered symmetry breaking and comet-tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations suggesting myosin-I acts as a repulsive force shaping the networks architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.
Score: 21.7, Published: 2024-02-09
DOI: 10.1101/2023.09.06.556521
The cell membrane must balance mechanical stability with fluidity to function as both a barrier and an organizational platform. Key to this balance is the thermodynamic ordering of lipids. Most Eukaryotes employ sterols, which are uniquely capable of modulating lipid order to decouple membrane stability from fluidity. Ancient sterol analogues known as hopanoids are found in many bacteria and are proposed as ancestral ordering lipids. The juxtaposition of sterols and hopanoids in extant organisms prompts us to ask why both pathways persist, especially in light of their convergent ability to order lipids. We reveal that both hopanoids and sterols order unsaturated phospholipids differently based on the position of double bonds in the phospholipids acyl chain. We find that cholesterol and diplopterols methyl group distributions lead to distinct effects on unsaturated lipids. In Mesoplasma florum, diplopterols constrained ordering capacity reduces membrane resistance to osmotic stress, unlike cholesterol. These findings suggest cholesterols broader lipid ordering ability may have facilitated the exploration of a more diverse lipidomic landscape in eukaryotic membranes.