FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.

Design of biologically active binary protein 2D materials.

Ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers6,7, but with the exception of a single lattice formed with flexible linkers8, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality9-12. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.

ERnet: a tool for the semantic segmentation and quantitative analysis of endoplasmic reticulum topology.

The ability to quantify structural changes of the endoplasmic reticulum (ER) is crucial for understanding the structure and function of this organelle. However, the rapid movement and complex topology of ER networks make this challenging. Here, we construct a state-of-the-art semantic segmentation method that we call ERnet for the automatic classification of sheet and tubular ER domains inside individual cells. Data are skeletonized and represented by connectivity graphs, enabling precise and efficient quantification of network connectivity. ERnet generates metrics on topology and integrity of ER structures and quantifies structural change in response to genetic or metabolic manipulation. We validate ERnet using data obtained by various ER-imaging methods from different cell types as well as ground truth images of synthetic ER structures. ERnet can be deployed in an automatic high-throughput and unbiased fashion and identifies subtle changes in ER phenotypes that may inform on disease progression and response to therapy.

Mutation of the ALS/FTD-associated RNA-binding protein FUS affects axonal development.

Aberrant condensation and localisation of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organisation and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signalling. To assess whether loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.Significance statement This study demonstrates that mutation of the ALS/FTD (amyotrophic lateral sclerosis/frontotemporal dementia)-associated RNA-binding protein Fused in Sarcoma (FUS) can result in changes in axonal development. These changes occur both axon-autonomously in cytoskeletal organisation during axon extension and context-dependently during axonal branching. This indicates pre-symptomatic, developmental changes in axonal organisation may occur in familial disease variants.

Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection.

Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.

Fluorescent Nanoparticles for Super-Resolution Imaging.

Super-resolution imaging techniques that overcome the diffraction limit of light have gained wide popularity for visualizing cellular structures with nanometric resolution. Following the pace of hardware developments, the availability of new fluorescent probes with superior properties is becoming ever more important. In this context, fluorescent nanoparticles (NPs) have attracted increasing attention as bright and photostable probes that address many shortcomings of traditional fluorescent probes. The use of NPs for super-resolution imaging is a recent development and this provides the focus for the current review. We give an overview of different super-resolution methods and discuss their demands on the properties of fluorescent NPs. We then review in detail the features, strengths, and weaknesses of each NP class to support these applications and provide examples from their utilization in various biological systems. Moreover, we provide an outlook on the future of the field and opportunities in material science for the development of probes for multiplexed subcellular imaging with nanometric resolution.

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended ß-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.

Single-Molecule Sizing through Nanocavity Confinement.

An approach relying on nanocavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nanocavity diffusional sizing (NDS), measures particle residence times within nanofluidic cavities to determine their hydrodynamic radii. Using theoretical modeling and simulations, we show that the residence time of particles within nanocavities above a critical time scale depends on the diffusion coefficient of the particle, which allows the estimation of the particle's size. We demonstrate this approach experimentally through the measurement of particle residence times within nanofluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates, and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential applications in nanobiotechnology, biophysics, and clinical diagnostics.

EAP45 association with budding HIV-1: Kinetics and domain requirements.

A number of viruses including HIV use the ESCRT system to bud from the infected cell. We have previously confirmed biochemically that ESCRT-II is involved in this process in HIV-1 and have defined the molecular domains that are important for this. Here, using SNAP-tag fluorescent labelling and both fixed and live cell imaging we show that the ESCRT-II component EAP45 colocalises with the HIV protein Gag at the plasma membrane in a temporal and quantitative manner, similar to that previously shown for ALIX and Gag. We show evidence that a proportion of EAP45 may be packaged within virions, and we confirm the importance of the N terminus of EAP45 and specifically the H0 domain in this process. By contrast, the Glue domain of EAP45 is more critical for recruitment during cytokinesis, emphasising that viruses have ways of recruiting cellular components that may be distinct from those used by some cellular processes. This raises the prospect of selective interference with the pathway to inhibit viral function while leaving cellular functions relatively unperturbed.

Molecular Mechanisms of Cationic Fusogenic Liposome Interactions with Bacterial Envelopes.

Although fusogenic liposomes offer a promising approach for the delivery of antibiotic payloads across the cell envelope of Gram-negative bacteria, there is still a limited understanding of the individual nanocarrier interactions with the bacterial target. Using super-resolution microscopy, we characterize the interaction dynamics of positively charged fusogenic liposomes with Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacteria. The liposomes merge with the outer membrane (OM) of Gram-negative bacteria, while attachment or lipid internalization is observed in Gram-positive cells. Employing total internal reflection fluorescence microscopy, we demonstrated liposome fusion with model supported lipid bilayers. For whole E. coli cells, however, we observed heterogeneous membrane integrations, primarily involving liposome attachment and hemifusion events. With increasing lipopolysaccharide length, the likelihood of full-fusion events was reduced. The integration of artificial lipids into the OM of Gram-negative cells led to membrane destabilization, resulting in decreased bacterial vitality, membrane detachment, and improved codelivery of vancomycin─an effective antibiotic against Gram-positive cells. These findings provide significant insights into the interactions of individual nanocarriers with bacterial envelopes at the single-cell level, uncovering effects that would be missed in bulk measurements. This highlights the importance of conducting single-particle and single-cell investigations to assess the performance of next-generation drug delivery platforms.

A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication.

Herpesviruses are large and complex viruses that have a long history of coevolution with their host species. One important factor in the virus-host interaction is the alteration of intracellular morphology during viral replication with critical implications for viral assembly. However, the details of this remodeling event are not well understood, in part because insufficient tools are available to deconstruct this highly heterogeneous process. To provide an accurate and reliable method of investigating the spatiotemporal dynamics of virus-induced changes to cellular architecture, we constructed a dual-fluorescent reporter virus that enabled us to classify four distinct stages in the infection cycle of herpes simplex virus-1 at the single cell level. This timestamping method can accurately track the infection cycle across a wide range of multiplicities of infection. We used high-resolution fluorescence microscopy analysis of cellular structures in live and fixed cells in concert with our reporter virus to generate a detailed and chronological overview of the spatial and temporal reorganization during viral replication. The highly orchestrated and striking relocation of many organelles around the compartments of secondary envelopment during transition from early to late gene expression suggests that the reshaping of these compartments is essential for virus assembly. We furthermore find that accumulation of HSV-1 capsids in the cytoplasm is accompanied by fragmentation of the Golgi apparatus with potential impact on the late steps of viral assembly. We anticipate that in the future similar tools can be systematically applied for the systems-level analysis of intracellular morphology during replication of other viruses.

Intracellular Aß42 Aggregation Leads to Cellular Thermogenesis.

The aggregation of Aß42 is a hallmark of Alzheimer's disease. It is still not known what the biochemical changes are inside a cell which will eventually lead to Aß42 aggregation. Thermogenesis has been associated with cellular stress, the latter of which may promote aggregation. We perform intracellular thermometry measurements using fluorescent polymeric thermometers to show that Aß42 aggregation in live cells leads to an increase in cell-averaged temperatures. This rise in temperature is mitigated upon treatment with an aggregation inhibitor of Aß42 and is independent of mitochondrial damage that can otherwise lead to thermogenesis. With this, we present a diagnostic assay which could be used to screen small-molecule inhibitors to amyloid proteins in physiologically relevant settings. To interpret our experimental observations and motivate the development of future models, we perform classical molecular dynamics of model Aß peptides to examine the factors that hinder thermal dissipation. We observe that this is controlled by the presence of ions in its surrounding environment, the morphology of the amyloid peptides, and the extent of its hydrogen-bonding interactions with water. We show that aggregation and heat retention by Aß peptides are favored under intracellular-mimicking ionic conditions, which could potentially promote thermogenesis. The latter will, in turn, trigger further nucleation events that accelerate disease progression.

Label-Free Characterization of Amyloids and Alpha-Synuclein Polymorphs by Exploiting Their Intrinsic Fluorescence Property.

Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of ß-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), ß-Lactoglobulin (ßLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and ßLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.

Nanoscale Features of Tunable Bacterial Outer Membrane Models Revealed by Correlative Microscopy.

The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system's tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein-protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.

A method for the fast and photon-efficient analysis of time-domain fluorescence lifetime image data over large dynamic ranges.

Fluorescence lifetime imaging (FLIM) allows the quantification of sub-cellular processes in situ, in living cells. A number of approaches have been developed to extract the lifetime from time-domain FLIM data, but they are often limited in terms of speed, photon efficiency, precision or the dynamic range of lifetimes they can measure. Here, we focus on one of the best performing methods in the field, the centre-of-mass method (CMM), that conveys advantages in terms of speed and photon efficiency over others. In this paper, however, we identify a loss of photon efficiency of CMM for short lifetimes when background noise is present. We subsequently present a new development and generalization of CMM that provides for the rapid and accurate extraction of fluorescence lifetime over a large lifetime dynamic range. We provide software tools to simulate, validate and analyse FLIM data sets and compare the performance of our approach against the standard CMM and the commonly employed least-square minimization (LSM) methods. Our method features a better photon efficiency than standard CMM and LSM and is robust in the presence of background noise. The algorithm is applicable to any time-domain FLIM data set.

Guided Assembly and Patterning of Intrinsically Fluorescent Amyloid Fibers with Long-Range Order.

Fibrillar amyloids exhibit a fascinating range of mechanical, optical, and electronic properties originating from their characteristic ß-sheet-rich structure. Harnessing these functionalities in practical applications has so far been hampered by a limited ability to control the amyloid self-assembly process at the macroscopic scale. Here, we use core-shell electrospinning with microconfinement to assemble amyloid-hybrid fibers, consisting of densely aggregated fibrillar amyloids stabilized by a polymer shell. Up to centimeter-long hybrid fibers with micrometer diameter can be arranged into aligned and ordered arrays and deposited onto substrates or produced as free-standing networks. Properties that are characteristic of amyloids, including their high elastic moduli and intrinsic fluorescence signature, are retained in the hybrid fiber cores, and we show that they fully persist through the macroscopic fiber patterns. Our findings suggest that microlevel confinement is key for the guided assembly of amyloids from monomeric proteins.

Advances in the Sensing and Treatment of Wound Biofilms.

Wound biofilms represent a particularly challenging problem in modern medicine. They are increasingly antibiotic resistant and can prevent the healing of chronic wounds. However, current treatment and diagnostic options are hampered by the complexity of the biofilm environment. In this review, we present new chemical avenues in biofilm sensors and new materials to treat wound biofilms, offering promise for better detection, chemical specificity, and biocompatibility. We briefly discuss existing methods for biofilm detection and focus on novel, sensor-based approaches that show promise for early, accurate detection of biofilm formation on wound sites and that can be translated to point-of-care settings. We then discuss technologies inspired by new materials for efficient biofilm eradication. We focus on ultrasound-induced microbubbles and nanomaterials that can both penetrate the biofilm and simultaneously carry active antimicrobials and discuss the benefits of those approaches in comparison to conventional methods.

Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid ß1-42 pathologies.

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-ß 1-42 (Aß42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

Advances in the study of organelle interactions and their role in neurodegenerative diseases enabled by super-resolution microscopy.

From the first illustrations of neuronal morphology by Ramón y Cajal to the recent three-dimensional reconstruction of synaptic connections, the development of modern neuroscience has greatly benefited from breakthroughs in imaging technology. This also applies specifically to the study of neurodegenerative diseases. Much of the research into these diseases relies on the direct visualisation of intracellular structures and their dynamics in degenerating neural cells, which cannot be fully resolved by diffraction-limited microscopes. Progress in the field has therefore been closely linked to the development of super-resolution imaging methods. Their application has greatly advanced our understanding of disease mechanisms, ranging from the structural progression of protein aggregates to defects in organelle morphology. Recent super-resolution studies have specifically implicated the disruption of inter-organelle interactions in multiple neurodegenerative diseases. In this article, we describe some of the key super-resolution techniques that have contributed to this field. We then discuss work to visualise changes in the structure and dynamics of organelles and associated dysfunctions. Finally, we consider what future developments in imaging technology may further our knowledge of these processes.