Biology subjects

Sanders, K.

Publications and source records attributed to Sanders, K..

At least 19 recordsLinked to original sources

bioRxiv2026

MaCADEMIA: a software toolbox for single-molecule fluorescence image quantification

Single-molecule localisation microscopy (SMLM) is a powerful tool in molecular and cellular biology and part of the broad umbrella of super-resolution microscopy. A distinct feature of SMLM, is its capacity for deep quantification, beyond just spatial information, to copy number, stoichiometry, molecular interaction and dynamics in living cells. The hardware required is increasingly straightforward to implement. However, complexity remains in the image analysis where much remains fragmented across specialised software tools. Here, we present MaCADEMIA (Matlab Computational Analysis of Diffusion, Emitter Molecular Intensity and Abundance), a simple yet powerful toolbox of MATLAB functions and associated documentation to enable straightforward extraction of quantitative single-molecule information. MaCADEMIA performs ensemble particle detection achieving localisation accuracies down to 0.5 pixels Root mean squared error (RMSE) across wide signal-to-noise (SNR) ranges on standardised particle tracking benchmarks, consistently matching or exceeding the performance of 14 leading methods. Our integrated workflow enables molecular counting and stoichiometry determination through stepwise photobleaching analysis, mobility measurement via mean squared displacement, colocalisaton analysis and absolute protein copy number quantification using three-dimensional cellular models.

bioinformatics↗

bioRxiv2026

A human lung organoid co-culture model of early bone sarcoma metastasis reveals contact-dependent epithelial remodeling at the metastatic interface

Lung metastasis drives mortality across cancer types, yet how infiltrating tumor cells remodel the lung epithelium to establish metastatic niches remains poorly understood. Here we establish MESCUL (MEtastatic Sarcoma Co-CULture), a co-culture platform combining human lung organoids with patient-derived bone sarcoma cells to model early tumor-lung epithelial interactions in a physiologically relevant 3D system. MESCUL reveals that direct tumor-epithelial contact induces rapid, reproducible lung epithelial remodeling across Ewing sarcoma (ES) and osteosarcoma (OS) models and multiple organoid donor backgrounds, which is contact-dependent and not recapitulated by paracrine signaling. Single-cell RNA sequencing identifies LIMES (Lung Interface Metastasis Signature), a shared transcriptional program encompassing focal adhesion assembly, matrix metalloprotease (MMP) upregulation, and emergence of a damage-associated transitional cell state, in both ES and OS. Mechanistically, tumor-derived fibronectin (FN1) engages epithelial integrin receptors to activate focal adhesion kinase (FAK), driving amphiregulin (AREG) induction and MMP-mediated remodeling; FN1 alone phenocopies this response, and FAK inhibition attenuates it, nominating the FN1-integrin-FAK-AREG axis as a candidate therapeutic vulnerability. The LIMES program, identified through the MESCUL co-culture model, is spatially confined to the tumor-lung interface in patient metastases of both ES and OS, as demonstrated by spatial transcriptomics across nine patients. Massons trichrome staining of matched patient sections reveals pronounced collagen deposition in the peri-tumoral lung parenchyma, consistent with LIMES acting upstream of a wound-healing cascade that proceeds to structural fibrotic remodeling in patient tissue. Together, these findings establish the lung epithelium as an active participant in metastatic colonization, characterize a pharmacologically targetable, spatially restricted epithelial remodeling response at the bone sarcoma-lung interface across OS and ES, and introduce MESCUL as a tractable 3D platform for investigating lung metastasis, with possible implications for tumor types beyond bone sarcomas.

cancer biology↗

bioRxiv2026

Topographically segregated mediodorsal-prefrontal loops exhibit distinct cue dynamics during learning and extinction

Using external cues to guide behavior is a core function that enables multiple aspects of cognition and attentional control, and deficits in this process are central to many theories of neuro-psychiatric, degenerative, and developmental disorders. Cue detection relies on the precise coordination of neural circuits, with the mediodorsal thalamus (MD) hypothesized to play a pivotal role in orchestrating the relay of cue-based associative information to the prefrontal cortex. The prefrontal cortex comprises multiple subregions, which are believed to differentially contribute to such associative cue-based behaviors. This regional specificity is likely seeded by projection-defined MD[->]PFC pathways, although the anatomical organization of these discrete channels and their dynamic roles in cue detection are still being defined. Here, we address this gap by combining anatomical circuit mapping of MD-PFC output pathways with in vivo calcium imaging during a cue-based reward conditioning task in mice. These experiments reveal that MD projections to distinct PFC subregions (prelimbic and anterior cingulate cortex) form topographically defined loops, that are characterized by unique patterns of activity across cue-reward learning. Using fiber photometry to monitor changes in calcium activity in axonal projections from the MD to the PFC, we show that during learning, MD projections to the prelimbic subregion are activated by cue presentation, and the dynamics of this activity remain stable across training days. In contrast, MD projections to the anterior cingulate exhibit a learning-dependent suppression of activity that predicts reward approach behavior in late training. Interestingly, the two pathways exhibit opposing activity patterns when the predictive validity of the cue is diminished by extinction training, suggesting distinct functional roles in detecting violations of learned contingencies. Together, these findings reveal previously unrecognized anatomical and functional distinctions within MD-PFC circuits and demonstrate that parallel thalamocortical pathways differentially support cue detection and behavioral flexibility. This work advances understanding of thalamocortical mechanisms underlying cue detection and may inform circuit-based approaches for treating cognitive dysfunction in psychiatric disorders.

neuroscience↗

bioRxiv2024

Discovery and characterization of stereodefined PMO-gapmers targeting tau

Antisense oligonucleotides (ASOs) are an important class of therapeutics to treat genetic diseases, and expansion of this modality to neurodegenerative disorders has been an active area of research. To realize chronic administration of ASO therapeutics to treat neurogenerative diseases, new chemical modifications improving activity and safety profile are still needed. Furthermore, it is highly desirable to develop a single stereopure ASO with defined activity and safety profile to avoid any efficacy and safety concerns due to the batch-to-batch variation in the composition of diastereomers. Herein, a stereopure PMO-gapmer was developed as a new construct to improve safety and stability by installing charge-neutral PMOs at the wing region and by fully controlling phosphorus stereochemistries. The developed stereopure PMO-gapmer construct was applied to the discovery of ASO candidates for the reduction of microtubule-associated protein tau (MAPT, tau). Sequence screening targeting MAPT followed by screening of optimal phosphorus stereochemistry identified stereopure development candidates. While evaluating the stereopure PMO-gapmers, we observed a dramatic difference in safety profile among stereoisomers in which only one phosphorus stereochemistry differs. These results further highlight the benefits of developing stereopure ASOs as safe and well-characterized candidates for clinical studies.

neuroscience↗

bioRxiv2023

Sinefungin, a natural nucleoside analog of S-adenosyl methionine, impairs the pathogenicity of Candida albicans

Candida albicans, an opportunistic fungal human pathogen, is a major threat to the healthcare system due to both infections in immunocompromised individuals and the emergence of antifungal resistance. Fungal infection caused by C. albicans, candidiasis, is a life-threatening condition in immunocompromised patients and the current treatments are mostly restricted to polyenes, azoles, and echinocandins. Use of these antifungals is limited by toxicity, drug-drug interactions, and the emergence of resistance, underscoring the importance of identifying novel therapeutic targets and the need for new treatment approaches. C. albicans can undergo a morphological transition from yeast to hyphae and this transition is central to C. albicans virulence. Here, we determine the impact of sinefungin, a natural nucleoside analog of S-adenosyl methionine, on the virulence of C. albicans strain SC5314 by evaluating treatment effects on the morphological transition, human epithelial cell adhesion, and biofilm formation. Our data indicate that sinefungin impairs pathogenic traits of C. albicans including hyphal lengthening, biofilm formation and the adhesion to the human epithelial cell lines, without adversely affecting human cells, therefore highlighting sinefungin as a potential avenue for therapeutic intervention. We determine that the formation of N6-methyladenosine (m6A) is particularly disturbed by sinefungin. More broadly, this study underscores the importance of considering the post-transcriptional control mechanisms of pathogenicity when designing therapeutic solutions to fungal infection.

biochemistry↗

bioRxiv2022

Substrate selectivity of the PRDM9 lysine methyltransferase domain.

Lysine methylation is a dynamic, post-translational mark that regulates the function of histone and non-histone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate non-histone proteins. In this work, we investigate the substrate selectivity of the lysine methyltransferase PRDM9 to identify both potential histone and non-histone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on non-histone proteins. Using lysine-oriented peptide (K-OPL) libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite {lambda}-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative non-histone substrates, which were tested by peptide spot array. Finally, PRDM9 methylation non-histone substrates were validated at the protein level by in vitro KMT assays on recombinant proteins. The selectivity profile of PRDM9 will be useful in identifying putative PRDM9 substrates in different cellular contexts, and future studies are required to determine whether PRDM9 methylates non-histone proteins in the context of meiotic recombination or cancer.

biochemistry↗

bioRxiv2022

Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius

Surface layers (S-layers) are resilient two-dimensional protein lattices that encapsulate many bacteria and most archaea. In archaea, S-layers usually form the only structural component of the cell wall and thus act as the final frontier between the cell and its environment. Therefore, S-layers are crucial for supporting microbial life. Notwithstanding their importance, little is known about archaeal S-layers at the atomic level. Here, we combined single particle cryo electron microscopy (cryoEM), cryo electron tomography (cryoET) and Alphafold2 predictions to generate an atomic model of the two-component S-layer of Sulfolobus acidocaldarius. The outer component of this S-layer (SlaA) is a flexible, highly glycosylated, and stable protein. Together with the inner and membrane-bound component (SlaB), they assemble into a porous and interwoven lattice. We hypothesize that jackknife-like conformational changes, as well as pH-induced alterations in the surface charge of SlaA, play important roles in S-layer assembly.

microbiology↗

bioRxiv2022

A comparison of six DNA extraction protocols for 16S, ITS, and shotgun metagenomic sequencing of microbial communities

Microbial communities contain a broad phylogenetic diversity of organisms, however the majority of methods center on describing bacteria and archaea. Fungi are important symbionts in many ecosystems, and are potentially important members of the human microbiome, beyond those that can cause disease. To expand our analysis of microbial communities to include fungal ITS data, we compared five candidate DNA extraction kits against our standardized protocol for describing bacteria and archaea using 16S rRNA gene amplicon- and shotgun metagenomics sequencing. We present results considering a diverse panel of host-associated and environmental sample types, and comparing the cost, processing time, well-to-well contamination, DNA yield, limit of detection, and microbial community composition among protocols. Across all criteria, we found the MagMAX Microbiome kit to perform best. The PowerSoil Pro kit performed comparably, but with increased cost per sample and overall processing time. The Zymo MagBead, NucleoMag Food, and Norgen Stool kits were included. Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_kit_testing). Methods summaryTo allow for downstream applications involving fungi in addition to bacteria and archaea, we compared five DNA extraction kits with our previously established, standardized protocol for extracting DNA for microbial community analysis. Across ten diverse sample types, we found one extraction kit to perform comparably or better than our standardized protocol. Our conclusion is based on per-sample comparisons of DNA yield, the number of quality-filtered sequences generated, the limit of detection of microbial cells, microbial community alpha-diversity, beta-diversity, and taxonomic composition, and extent of well-to-well contamination.

microbiology↗

bioRxiv2022

Sentinel Cards Provide Practical SARS-CoV-2 Monitoring in School Settings

Accurate, high-resolution environmental monitoring of SARS-CoV-2 traces indoors through sentinel cards is a promising approach to help students safely return to in-person learning. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface types, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution, but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, wet wipes, RNase Away) at three different viral loads: "high" (4 x 104 GE/mL), "medium" (1 x 104 GE/mL), and "low" (2.5 x 103 GE/mL). RNAse Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. ImportanceBecause SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are persistent signal or new infectious cases, and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.

bioinformatics↗

bioRxiv2021

Circulating microRNAs as noninvasive biomarkers for canine Cushing's syndrome

Canine Cushings syndrome (hypercortisolism) can be caused by a pituitary tumor (pituitary-dependent hypercortisolism; PDH) or a cortisol-secreting adrenocortical tumor (csACT). For both cases, noninvasive biomarkers that could pre-operatively predict the risk of recurrence after surgery would greatly impact clinical decision making. The aim of this study was to determine whether circulating microRNAs (miRNAs) can be used as noninvasive biomarkers for canine Cushings syndrome. After a pilot study with 40 miRNAs in blood samples of healthy dogs (n = 3), dogs with PDH (n = 3) and dogs with a csACT (n = 4), we selected a total of 20 miRNAs for the definitive study. In the definitive study, these 20 miRNAs were analyzed in blood samples of healthy dogs (n = 6), dogs with PDH (n = 19, pre-and post-operative samples) and dogs with a csACT (n = 26, pre-operative samples). In dogs with PDH, six miRNAs (miR-122-5p, miR-126-5p, miR-141-3p, miR-222-3p, miR-375-3p and miR-483-3p) were differentially expressed compared to healthy dogs. Of one miRNA, miR-122-5p, the expression levels did not overlap between healthy dogs and dogs with PDH (p = 2.9x10-4), significantly decreased after hypophysectomy (p = 0.013), and were significantly higher (p = 0.017) in dogs with recurrence (n = 3) than in dogs without recurrence for at least one year after hypophysectomy (n = 7). In dogs with csACTs, two miRNAs (miR-483-3p and miR-223-3p) were differentially expressed compared to healthy dogs. Additionally, miR-141-3p was expressed significantly lower (p = 0.009) in dogs with csACTs that had a histopathological Utrecht score of [≥] 11 compared to those with a score of < 11. These results indicate that circulating miRNAs have the potential to be noninvasive biomarkers in dogs with Cushings syndrome that may contribute to clinical decision-making.

cancer biology↗

bioRxiv2021

CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage

The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 [A] resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.

biophysics↗

bioRxiv2021

Multi-omics profiling of Earth's biomes reveals that microbial and metabolite composition are shaped by the environment

As our understanding of the structure and diversity of the microbial world grows, interpreting its function is of critical interest for understanding and managing the many systems microbes influence. Despite advances in sequencing, lack of standardization challenges comparisons among studies that could provide insight into the structure and function of microbial communities across multiple habitats on a planetary scale. Technical variation among distinct studies without proper standardization of approaches prevents robust meta-analysis. Here, we present a multi-omics, meta-analysis of a novel, diverse set of microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry), centering our description on relationships and co-occurrences of microbially-related metabolites and microbial taxa across environments. Standardized protocols and analytical methods for characterizing microbial communities, including assessment of molecular diversity using untargeted metabolomics, facilitate identification of shared microbial and metabolite features, permitting us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of a community resource that will become more valuable with time. To provide examples of applying this database, we outline important ecological questions that can be addressed, and test the hypotheses that every microbe and metabolite is everywhere, but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment. The relative abundances of microbially-related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner, and highlight the power of certain chemistry - in particular terpenoids - in distinguishing Earths environments.

ecology↗

bioRxiv2020

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

One goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols. Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX). Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

ecology↗

bioRxiv2020

Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the colon

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contributing to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of SMCs using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+ transients [~]15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+ firing patterns and drive smooth muscle activity and overall colonic contractions. SynopsisHow Ca2+ signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is unknown. This study shows how ICC modulate colonic motility via complex Ca2+ signaling and defines Ca2+ transients sources using optogenetic techniques.

cell biology↗

bioRxiv2020

Optical spike detection and connectivity analysis with a far-red voltage-sensitive fluorophore reveals changes to network connectivity in development and disease

The ability to optically record dynamics of neuronal membrane potential promises to revolutionize our understanding of neurobiology. In this study, we show that the far-red voltage sensitive fluorophore, Berkeley Red Sensor of Transmembrane potential -1, or BeRST 1, can be used to monitor neuronal membrane potential changes across dozens of neurons at a sampling rate of 500 Hz. Notably, voltage imaging with BeRST 1 can be implemented with affordable, commercially available illumination sources, optics, and detectors. BeRST 1 is well-tolerated in cultures of rat hippocampal neurons and provides exceptional optical recording fidelity, as judged by dual fluorescence imaging and patch-clamp electrophysiology. We developed a semi-automated spike-picking program to reduce user bias when calling action potentials and used this in conjunction with BeRST 1 to develop an optical spike and connectivity analysis workflow (OSCA) for high-throughput dissection of neuronal activity dynamics in development and disease. The high temporal resolution of BeRST 1 enables dissection of firing rate changes in response to acute, pharmacological interventions with commonly used inhibitors like gabazine and picrotoxin. Over longer periods of time, BeRST 1 also tracks chronic perturbations to neurons exposed to amyloid beta (A{beta}1-42), revealing modest changes to spiking frequency but profound changes to overall network connectivity. Finally, we use OSCA to track changes in neuronal connectivity during development, providing a functional readout of network assembly. We envision that use of BeRST 1 and OSCA described here will be of use to the broad neuroscience community. Significance StatementOptical methods to visualize membrane potential dynamics provide a powerful complement to Ca2+ imaging, patch clamp electrophysiology, and multi-electrode array recordings. However, modern voltage imaging strategies often require complicated optics, custom-built microscopes, or genetic manipulations that are impractical outside of a subset of model organisms. Here, we describe the use of Berkeley Red Sensor of Transmembrane potential, or BeRST 1, a far-red voltage-sensitive fluorophore that can directly visualize membrane potential changes with millisecond resolution across dozens of neurons. Using only commercially available components, voltage imaging with BeRST 1 reveals profound changes in neuronal connectivity during development, exposes changes to firing rate during acute pharmacological perturbation, and illuminates substantial increases in network connectivity in response to chronic exposure to amyloid beta.

neuroscience↗

bioRxiv2020

Imaging voltage in complete neuronal networks within patterned microislands reveals preferential wiring of excitatory hippocampal neurons

Voltage imaging with fluorescent dyes affords the opportunity to map neuronal activity in both time and space. One limitation to imaging is the inability to image complete neuronal networks: some fraction of cells remains outside of the observation window. Here, we combine voltage imaging, post hoc immunocytochemistry, and patterned microisland hippocampal culture to provide imaging of complete neuronal networks. The patterned microislands completely fill the field of view of our high-speed (500 Hz) camera, enabling reconstruction of the spiking patterns of every single neuron in the network. Cultures raised on microislands develop similarly to neurons grown on coverslips and display similar composition of inhibitory and excitatory cell types. The principal excitatory cell types (CA1, CA3, and dentate granule cells, or DGC) are also present in similar proportions in both preparations. We calculate the likelihood that action potential firing in one neuron to trigger action potential firing in a downstream neuron in a spontaneously active network to construct a functional connection map of these neuronal ensembles. Importantly, this functional map indicates preferential connectivity between DGC and CA3 neurons and between CA3 and CA1 neurons, mimicking the neuronal circuitry of the intact hippocampus. We envision that patterned microislands, in combination with voltage imaging and methods to classify cell types, will be a powerful method for exploring neuronal function in both healthy and disease states. Additionally, because the entire neuronal network is sampled simultaneously, this strategy has the power to go further, revealing all functional connections between all cell types. Significance StatementIn vitro model systems provide unsurpassed control and access for exploring the molecular and cellular details of neurobiology. We developed a patterned microisland system for culturing rat hippocampal neurons that recapitulates the features of bulk hippocampal cultures, but with the added benefit of allowing access to high-speed imaging of entire neuronal ensembles using voltage imaging. By using far-red voltage-sensitive fluorophores, we map the functional connections across all cells in the neuronal ensemble, revealing that several important functional synapses present in the intact hippocampus are recapitulated in this microisland system. We envision the methods described here will be a powerful complement to ongoing research into basic neurobiological mechanisms and the search for therapies to treat diseases arising from their dysfunction.

neuroscience↗

bioRxiv2019

A new twist on bacterial motility - Two distinct type IV pili revealed by cryoEM

Many bacteria express flexible protein filaments on their surface that enable a variety of important cellular functions. Type IV pili are examples of such filaments and are comprised of a helical assembly of repeating pilin subunits. Type IV pili are involved in motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). They are therefore powerful structures that enable bacterial proliferation and genetic adaptation, potentially leading to the development of pathogenicity and antibiotic resistance. They are also targets for drug development.\n\nBy a complement of experimental approaches, we show that the bacterium Thermus thermophilus produces two different forms of type IV pilus. We have determined the structures of both and built atomic models. The structures answer key unresolved questions regarding the molecular architecture of type IV pili and identify a new type of pilin. We also delineate the roles of the two filaments in promoting twitching and natural transformation.

biophysics↗

bioRxiv2019

Architecture and modular assembly of the Sulfolobus S-layers revealed by electron cryo-tomography

Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic and mechanical stability, the formation of a semi-permeable protective barrier around the cell, cell-cell interaction, as well as surface adhesion. Despite the central importance of the S-layer for archaeal life, their three-dimensional architecture is still poorly understood. Here we present the first detailed 3D electron cryo-microscopy maps of archaeal S-layers from three different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the two subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.

microbiology↗