Localization atomic force microscopy.

Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.

BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes.

Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4K20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structural and single molecule imaging approaches. We found that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed- atomic force microscopy (HS-AFM) to monitor how the BRCA1-BARD1 complex recognises chromatin in real time, we saw a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiquitin ligase catalytic activity. Multivalent interactions across nucleosomes explain how BRCA1-BARD1 can recognise chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.

Innate and adaptive immune response of Rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri.

Rainbow trout is an important fish species for Peruvian artisanal aquaculture, comprising over 60 % of the total aquaculture production. However, their industry has been highly affected by several bacterial agents such as Yersinia ruckeri. This pathogen is the causative agent of Enteric Redmouth Disease, and causes high mortality in fingerlings and chronic infection in adult rainbow trout. To date, the immune response of rainbow trout against Y. ruckeri has been well studied in laboratory-controlled infection studies (i.e. intraperitoneal infection, bath immersion), however, the immune response during natural infection has not been explored. To address this, in this study, 35 clinically healthy O. mykiss without evidence of lesions or changes in behavior and 32 rainbow trout naturally infected by Y. ruckeri, were collected from semi-intensive fish farms located in the Central Highlands of Peru. To evaluate the effect on the immune response, RT-qPCR, western blotting, and ELISA were conducted using head kidney, spleen, and skin tissues to evaluate the relative gene expression and protein levels. Our results show a significant increase in the expression of the pro-inflammatory cytokines il1b, tnfa, and il6, as well as ifng in all three tissues, as well as increases in IL-1ß and IFN-γ protein levels. The endogenous pathway of antigen presentation showed to play a key role in defense against Y. ruckeri, due to the upregulation of mhc-I, tapasin, and b2m transcripts, and the significant increase of Tapasin protein levels in infected rainbow trout. None of the genes associated with the exogenous pathway of antigen presentation showed a significant increase in infected fish, suggesting that this pathway is not involved in the response against this intracellular pathogen. Finally, the transcripts of immunoglobulins IgM and IgT did not show a modulation, nor were the protein levels evaluated in this study.

A machine learning analysis of drought and rural population change on the North American Great Plains since the 1970s.

Machine learning techniques have to date not been widely used in population-environment research, but represent a promising tool for identifying relationships between environmental variables and population outcomes. They may be particularly useful for instances where the nature of the relationship is not obvious or not easily detected using other methods, or where the relationship potentially varies across spatial scales within a given study unit. Machine learning techniques may also help the researcher identify the relative strength of influence of specific variables within a larger set of interacting ones, and so provide a useful methodological approach for exploratory research. In this study, we use machine learning techniques in the form of random forest and regression tree analyses to look for possible connections between drought and rural population loss on the North American Great Plains between 1970 and 2020. In doing so, we analyzed four decades of population count data (at county-size spatial scales), monthly climate data, and Palmer Drought Severity Index scores for Canada and the USA at multiple spatial scales (regional, sub-regional, national, and county/census division levels), along with county level irrigation data. We found that in some parts of Saskatchewan and the Dakotas - particularly those areas that fall within more temperate/less arid ecological sub-regions - drought conditions in the middle years of the 1970s had a significant association with rural population losses. A similar but weaker association was identified in a small cluster of North Dakota counties in the 1990s. Our models detected few links between drought and rural population loss in other decades or in other parts of the Great Plains. Based on R-squared results, models for US portions of the Plains generally exhibited stronger drought-population loss associations than did Canadian portions, and temperate ecological sub-regions exhibited stronger associations than did more arid sub-regions. Irrigation rates showed no significant influence on population loss. This article focuses on describing the methodological steps, considerations, and benefits of employing this type of machine learning approach to investigating connections between drought and rural population change. Supplementary Information: The online version contains supplementary material available at 10.1007/s11111-022-00399-9.

Immune stimulation of rainbow trout reveals divergent regulation of MH class II-associated invariant chain isoforms.

Major histocompatibility complex (MHC) class II-associated invariant chain is a chaperone responsible for targeting the MHC class II dimer to the endocytic pathway, thus enabling the loading of exogenous antigens onto the MHC class II receptor. In the current study, in vivo and in vitro methods were used to investigate the regulation of the rainbow trout invariant chain proteins S25-7 and INVX, upon immune system activation. Whole rainbow trout and the macrophage/monocyte-like cell line RTS11 were treated with PMA at concentrations shown to induce IL-1ß transcripts and homotypic aggregation of RTS11. S25-7 transcript levels remained unchanged in the gill, spleen, and liver and were found to be significantly decreased in head kidney beginning 24 h post-stimulation. Meanwhile, INVX transcript levels remained unchanged in all tissues studied. Both S25-7 and INVX proteins were produced in gill and spleen tissues but their expression was unaffected by immune system stimulation. Surprisingly, neither INVX nor S25-7 protein was detected in the secondary immune organ, the head kidney. Analysis of RTS11 cultures demonstrated that both INVX and S25-7 transcript levels significantly increased at 96 h and 120 h following PMA stimulation before returning to control levels at 168 h. Meanwhile, at the protein level in RTS11, S25-7 remained unchanged while INVX had a significant decrease at 168 h post-stimulation. These results indicate that neither INVX nor S25-7 is upregulated upon immune system activation; thus, teleosts have evolved a system of immune regulation that is different than that found in mammals.

Visualization of diffusion limited antimicrobial peptide attack on supported lipid membranes.

Understanding the mechanism of action of antimicrobial peptides (AMP) is fundamental to the development and design of peptide based antimicrobials. Utilizing fast-scan atomic force microscopy (AFM) we detail the attack of an AMP on both prototypical prokaryotic (DOPC:DOPG) and eukaryotic (DOPC:DOPE) model lipid membranes on the nanoscale and in real time. Previously shown to have a favourable therapeutic index, we study Smp43, an AMP with a helical-hinge-helical topology isolated from the venom of the North African scorpion Scorpio maurus palmatus. We observe the dynamic formation of highly branched defects being supported by 2D diffusion models and further experimental data from liposome leakage assays and quartz crystal microbalance-dissipation (QCM-D) analysis, we propose that Smp43 disrupts these membranes via a common mechanism, which we have termed 'diffusion limited disruption' that encompasses elements of both the carpet model and the expanding pore mechanism.

Phospholipid dependent mechanism of smp24, an α-helical antimicrobial peptide from scorpion venom.

Determining the mechanism of action of antimicrobial peptides (AMPs) is critical if they are to be developed into the clinical setting. In recent years high resolution techniques such as atomic force microscopy (AFM) have increasingly been utilised to determine AMP mechanism of action on planar lipid bilayers and live bacteria. Here we present the biophysical characterisation of a prototypical AMP from the venom of the North African scorpion Scorpio maurus palmatus termed Smp24. Smp24 is an amphipathic helical peptide containing 24 residues with a charge of +3 and exhibits both antimicrobial and cytotoxic activity and we aim to elucidate the mechanism of action of this peptide on both membrane systems. Using AFM, quartz crystal microbalance-dissipation (QCM-D) and liposomal leakage assays the effect of Smp24 on prototypical synthetic prokaryotic (DOPG:DOPC) and eukaryotic (DOPE:DOPC) membranes has been determined. Our data points to a toroidal pore mechanism against the prokaryotic like membrane whilst the formation of hexagonal phase non-lamellar phase structures is seen in eukaryotic like membrane. Also, phase segregation is observed against the eukaryotic membrane and this study provides direct evidence of the same peptide having multiple mechanisms of action depending on the membrane lipid composition.

Layer-by-Layer Assembly of Supported Lipid Bilayer Poly-L-Lysine Multilayers.

Multilayer lipid membranes perform many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae), and sequential processing (golgi cisternae). Here we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic force microscopy (AFM) for up to six lipid bilayers. By varying buffer pH and PLL chain length, we show that longer chains (≥300 kDa) at pH 9.0 form thicker polymer supported multilayers, while at low pH and shorter length PLL, we create close packed layers (average lipid bilayers separations of 2.8 and 0.8 nm, respectively). Fluorescence recovery after photobleaching (FRAP) and AFM were used to show that the diffusion of lipid and three different membrane proteins in the multilayered membranes has little dependence on lipid stack number or separation between membranes. These approaches provide a straightforward route to creating the complex membrane structures that are found throughout nature, allowing possible applications in areas such as energy production and biosensing while developing our understanding of the biological processes at play.

New poly(amino acid methacrylate) brush supports the formation of well-defined lipid membranes.

A novel poly(amino acid methacrylate) brush comprising zwitterionic cysteine groups (PCysMA) was utilized as a support for lipid bilayers. The polymer brush provides a 12-nm-thick cushion between the underlying hard support and the aqueous phase. At neutral pH, the zeta potential of the PCysMA brush was ∼-10 mV. Cationic vesicles containing >25% DOTAP were found to form a homogeneous lipid bilayer, as determined by a combination of surface analytical techniques. The lipid mobility as measured by FRAP (fluorescence recovery after photobleaching) gave diffusion coefficients of ∼1.5 µm(2) s(-1), which are comparable to those observed for lipid bilayers on glass substrates.

Diffusion in low-dimensional lipid membranes.

The diffusion behavior of biological components in cellular membranes is vital to the function of cells. By collapsing the complexity of planar 2D membranes down to one dimension, fundamental investigations of bimolecular behavior become possible in one dimension. Here we develop lipid nanolithography methods to produce membranes, under fluid, with widths as low as 6 nm but extending to microns in length. We find reduced lipid mobility, as the width is reduced below 50 nm, suggesting different lipid packing in the vicinity of boundaries. The insertion of a membrane protein, M2, into these systems, allowed characterization of protein diffusion using high-speed AFM to demonstrate the first membrane protein 1D random walk. These quasi-1D lipid bilayers are ideal for testing and understanding fundamental concepts about the roles of dimensionality and size on physical properties of membranes from energy transfer to lipid packing.

Self-assembly of actin scaffolds on lipid microbubbles.

Microbubbles offer unique properties as combined carriers of therapeutic payloads and diagnostic agents. Here we report on the development of novel microbubble architectures that in addition to the usual lipid shell have an actin cytoskeletal cortex assembled on their exterior. We show, using atomic force microscopy that this biomimetic coating creates a thin mesh that allows tuning of the mechanical properties of microbubbles and that the nature of actin assembly is determined by the fluidity of the lipid layer. Further, we show that it is possible to attach payloads and targeting-ligands to the actin scaffold. Resistance to gas permeation showed that the additional actin layer reduces gas diffusion across the shell and thus increases bubble lifetime. This study demonstrates a one step method to creating more complex microbubble architectures, which would be capable of further modification and tuning through the inclusion of actin binding proteins.

NanoLocz: Image Analysis Platform for AFM, High-Speed AFM, and Localization AFM.

Atomic Force Microscopy (AFM), High-Speed AFM (HS-AFM) simulation AFM, and Localization AFM (LAFM) enable the study of molecules and surfaces with increasingly higher spatiotemporal resolution. However, effective and rapid analysis of the images and movies produced by these techniques can be challenging, often requiring the use of multiple image processing software applications and scripts. Here, NanoLocz, an open-source solution that offers advanced analysis capabilities for the AFM community, is presented. Integration and continued development of AFM analysis tools is essential to improve access to data, increase throughput, and open new analysis opportunities. NanoLocz efficiently leverages the rich data AFM has to offer by incorporating and combining existing and newly developed analysis methods for AFM, HS-AFM, simulation AFM, and LAFM seamlessly. It facilitates and streamlines AFM analysis workflows from import of raw data, through to various analysis workflows. Here, the study demonstrates the capabilities of NanoLocz and the new methods it enables including single-molecule LAFM, time-resolved LAFM, and simulation LAFM.

Actin assembly at model-supported lipid bilayers.

We report on the use of supported lipid bilayers to reveal dynamics of actin polymerization from a nonpolymerizing subphase via cationic phospholipids. Using varying fractions of charged lipid, lipid mobility, and buffer conditions, we show that dynamics at the nanoscale can be used to control the self-assembly of these structures. In the case of fluid-phase lipid bilayers, the actin adsorbs to form a uniform two-dimensional layer with complete surface coverage whereas gel-phase bilayers induce a network of randomly oriented actin filaments, of lower coverage. Reducing the pH increased the polymerization rate, the number of nucleation events, and the total coverage of actin. A model of the adsorption/diffusion process is developed to provide a description of the experimental data and shows that, in the case of fluid-phase bilayers, polymerization arises equally due to the adsorption and diffusion of surface-bound monomers and the addition of monomers directly from the solution phase. In contrast, in the case of gel-phase bilayers, polymerization is dominated by the addition of monomers from solution. In both cases, the filaments are stable for long times even when the G-actin is removed from the supernatant-making this a practical approach for creating stable lipid-actin systems via self-assembly.

Surface material of acoustic transmitters influences the inflammatory response of rainbow trout (Oncorhynchus mykiss) during long-term implantation.

Telemetry tags are a widely used technology for tracking animals that are difficult to observe in their natural environment. This technology has been increasingly used to monitor and study populations of high value salmonid species in Canadian waters. This study expands on a previous study of the impacts of tag implantation on the immune system of Rainbow Trout (Oncorhynchus mykiss). Pro-inflammatory cytokines and protein level markers were examined in fish that underwent peritoneal implantation of three tag types and compared to a sham surgery control group. The different materials on the surface of the tags showed differential immune induction extending over a two-month period. This included peritoneal total protein, IL-1ß protein, the immunoglobulins IgT and IgM, as well as pro-inflammatory transcripts in the spleen. These results are suggestive of a prolonged, costly foreign body response which may be differentially induced by the different types of tag coating, with ceramic tags being least immunogenic. Examining tag impacts at the level of the immune system will facilitate the development of more biocompatible tags which will improve data fidelity. This will support more effective strategies for the management of fisheries resources.

Expression of Interleukin-1ß protein in vitro,exvivo and in vivo salmonid models.

Interleukin-1ß (IL-1ß) is one of the first cytokines expressed during immune responses, and its levels are affected by many factors, including stress. To date, it has only been possible to measure IL-1ß transcript (mRNA) expression quantitatively in fish using qPCR. This is because previous studies that measured IL-1ß protein concentrations in these taxa used western blotting, which only provides qualitative data. To advance our knowledge of fish IL-1ß biology, and because post-translational processing plays a critical role in the activation of this molecule, we developed a quantitative enzyme-linked immunosorbent assay (ELISA) to accurately measure the concentration of IL-1ß protein in several cell cultures and in vivo in salmonids. We compared changes in IL-1ß protein levels to the expression of its mRNA. The developed ELISA was quite sensitive and has a detection limit of 12.5 pg/mL. The tools developed, and information generated through this research, will allow for a more accurate and complete understanding of IL-1ß's role in the immune response of salmonids.The assay described here has the potential to significantly advance our ability to assess fish health and immune status.

The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture.

The Bunyavirales order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies. IMPORTANCE Bunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for humans are not available. The bunyavirus genome is wrapped up by the nucleoprotein (NP) and interacts with the viral polymerase, forming a ribonucleoprotein (RNP). This is the only form of the genome active for viral replication and assembly. However, until now how NPs are organized within an RNP was not known for any orthobunyavirus. Here, we purified RNPs from the prototypical orthobunyavirus, Bunyamwera virus, and employed microscopy approaches to show that the NP portion of the RNP was helical. We then combined our helical average with the known structure of an NP monomer, generating a pseudo-atomic model of this region. This arrangement allowed the RNPs to be highly flexible, which was critical for several stages of the viral replication cycle, such as segment circularization.

Tuning the rate of aggregation of hIAPP into amyloid using small-molecule modulators of assembly.

Human islet amyloid polypeptide (hIAPP) self-assembles into amyloid fibrils which deposit in pancreatic islets of type 2 diabetes (T2D) patients. Here, we applied chemical kinetics to study the mechanism of amyloid assembly of wild-type hIAPP and its more amyloidogenic natural variant S20G. We show that the aggregation of both peptides involves primary nucleation, secondary nucleation and elongation. We also report the discovery of two structurally distinct small-molecule modulators of hIAPP assembly, one delaying the aggregation of wt hIAPP, but not S20G; while the other enhances the rate of aggregation of both variants at substoichiometric concentrations. Investigation into the inhibition mechanism(s) using chemical kinetics, native mass spectrometry, fluorescence titration, SPR and NMR revealed that the inhibitor retards primary nucleation, secondary nucleation and elongation, by binding peptide monomers. By contrast, the accelerator predominantly interacts with species formed in the lag phase. These compounds represent useful chemical tools to study hIAPP aggregation and may serve as promising starting-points for the development of therapeutics for T2D.

Single residue modulators of amyloid formation in the N-terminal P1-region of α-synuclein.

Alpha-synuclein (αSyn) is a protein involved in neurodegenerative disorders including Parkinson's disease. Amyloid formation of αSyn can be modulated by the 'P1 region' (residues 36-42). Here, mutational studies of P1 reveal that Y39A and S42A extend the lag-phase of αSyn amyloid formation in vitro and rescue amyloid-associated cytotoxicity in C. elegans. Additionally, L38I αSyn forms amyloid fibrils more rapidly than WT, L38A has no effect, but L38M does not form amyloid fibrils in vitro and protects from proteotoxicity. Swapping the sequence of the two residues that differ in the P1 region of the paralogue γSyn to those of αSyn did not enhance fibril formation for γSyn. Peptide binding experiments using NMR showed that P1 synergises with residues in the NAC and C-terminal regions to initiate aggregation. The remarkable specificity of the interactions that control αSyn amyloid formation, identifies this region as a potential target for therapeutics, despite their weak and transient nature.

Structural dynamics of channels and transporters by high-speed atomic force microscopy.

Channels and transporters are vital for transmembrane transport of ions and solutes, and also of larger compounds such as lipids and macromolecules. Therefore, they are crucial in many biological processes such as sensing, signal transduction, and the regulation of the distribution of molecules. Dysfunctions of these membrane proteins are associated to numerous diseases, and their interaction with drugs is critical in medicine. Understanding the behavior of channels and transporters requires structural and dynamic information to decipher the molecular mechanisms underlying their function. High-Speed Atomic Force Microscopy (HS-AFM) now allows the study of single transmembrane channels and transporters in action under physiological conditions, i.e., at ambient temperature and pressure, in physiological buffer and in a membrane, and in a most direct, label-free manner. In this chapter, we discuss the HS-AFM sample preparation, application, and data analysis protocols to study the structural and conformational dynamics of membrane-embedded channels and transporters.

Correlation of membrane protein conformational and functional dynamics.

Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the ß-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.