Biology subjects

Kaplan, M.

Publications and source records attributed to Kaplan, M..

At least 19 recordsLinked to original sources

bioRxiv2026

An Expanded Lpt Pathway Couples Surface Lipoprotein Export to Outer Membrane Vesicle Biogenesis in Bacteroides

The ecological dominance of Bacteroidota in the human gut microbiota derives from their exceptional capacity to metabolize diverse dietary and host-derived glycans. This ability relies on surface-exposed lipoproteins (SLPs), which are displayed on the bacterial surface and selectively packaged into outer membrane vesicles (OMVs). Although SLP export and OMV biogenesis are tightly coupled, the mechanism linking these processes has remained unknown. Here, we identify LptZ, a previously uncharacterized protein, as a critical link between SLP surface translocation and OMV biogenesis. Cryo-electron tomography of the lptZ mutant revealed chains of vesicles tethered to the outer membrane, offering a rare glimpse into an arrested stage of OMV formation in which vesicle fission is impaired. Proteomic analysis further showed that, in the absence of LptZ, a subset of SLPs fails to reach the cell surface and instead accumulates at the inner membrane. Unexpectedly, LptZ associates with the lipopolysaccharide transport (Lpt) machinery and repurposes it for SLP translocation. Crosslinking experiments revealed selective associations between multiple SLPs and Lpt proteins, providing mechanistic evidence for the direct involvement of the Lpt machinery in SLP trafficking. We further show that the Bacteroides Lpt machinery contains previously unrecognized components, revealing an unanticipated level of specialization relative to canonical Lpt systems. Together, our findings uncover a previously unknown protein export pathway, expand the functional role of the Lpt machinery, and establish a direct mechanistic link between SLP translocation and OMV biogenesis in Bacteroidota.

microbiology↗

bioRxiv2026

A Reduced Mechanistic Model for Aquatic Decomposition of a Human Body

Estimating the postmortem submersion interval (PMSI) remains challenging due to complex biological and environmental interactions during aquatic decomposition. This study proposes a reduced mechanistic ordinary differential equation (ODE) model linking tissue degradation, microbial activity, and dissolved oxygen dynamics. Calibrated against the total aquatic decomposition score (TADS) trajectory for Northern Adriatic Sea cases, the model preserves a strictly monotone TADS--time relationship, ensuring unique numerical inversion from observed TADS to PMSI. Synthetic observation experiments evaluated how data selection influences parameter recovery, practical identifiability, and robustness under measurement uncertainty. Microbial measurements improved parameter recovery for microbial dynamics, whereas dissolved oxygen measurements constrained oxygen-consumption and renewal processes. Combining both types of observation optimized the recovery of coupled parameters. Trajectory reconstruction, profile likelihood analysis, boundary-hit frequencies, and Monte Carlo simulations confirmed these findings. Importantly, while all observation strategies reproduced TADS with comparable accuracy, they differed substantially in recovering underlying biological parameters and latent-state dynamics. Thus, accurately reproducing decomposition scores alone is insufficient for reliable parameter inference. This framework provides a transparent and biologically interpretable foundation for the mechanistic estimation of PMSI. Future applications to case-level and multi-site datasets may enable site-specific recalibration and improved predictive evaluation across diverse aquatic environments.

systems biology↗

bioRxiv2026

Bacterial nanotubes extend from a tubeosome organelle to establish an intercellular bridge

Bacterial nanotubes are widespread intercellular membranous bridges mediating molecular exchange among neighboring cells, yet their formation and dynamics remain largely unexplored. By combining live-cell imaging with in situ cryo-electron tomography, we describe nanotube biogenesis in the Gram-positive Bacillus subtilis. Nanotubes form elaborate, highly dynamic networks, with extending nanotubes exhibiting multidirectional movement toward nearby cells, repeatedly scanning their surface. Ultimately, nanotubes penetrate through the recipient cell wall to establish an intercellular bridge. Strikingly, nanotubes emerge within milliseconds from lipid-enriched sites representing a previously unrecognized membranous organelle, termed tubeosome, located in an unusual pseudo-periplasmic space beneath the cell wall. We present evidence that tubeosomes comprise proto-nanotube reservoirs that unfold to project nanotubes. Our study uncovers the tubeosome as a novel bacterial organelle and provides the first real-time visualization of intercellular bridge formation. As connecting nanotubes are found across bacteria, archaea, and eukaryotes, this mechanism may reflect a broadly conserved strategy for cellular communication.

microbiology↗

bioRxiv2026

Diel remodeling and cellular integration of the nitroplast

Nitrogen-fixing eukaryotes were not believed to exist in nature until the recent discovery of a N2-fixing organelle, or nitroplast, in the marine microalga Braarudosphaera bigelowii. This nitroplast (formerly known as UCYN-A2) has long been recognized as key cyanobacterial contributor to global oceanic N2 fixation. However, how this novel organelle is integrated and regulated within the architecture of a eukaryotic cell remains unclear. Here, we combine multiscale volumetric imaging with cryo-electron tomography to resolve the native architecture, cellular integration, and diel remodeling of the nitroplast in cultured and environmental cells. We find that the nitroplast occupies up to 10% of the cell volume and exhibits close interfaces with multiple host organelles through membrane contact sites, while integration of this metabolically demanding compartment does not disrupt global scaling of host organelles. Interestingly, the chloroplast-to-nitroplast volume ratio is conserved across distinct life stages. Cryo-electron tomography reveals that the nitroplast retains a reinforced four-layer cyanobacterial envelope and is additionally surrounded by two host-derived layers that remodel across the day-night cycle. During daytime N2 fixation, these host-derived barriers become locally discontinuous and the organelle interface becomes enriched with two distinct vesicle populations. Our findings suggest that dynamic control of organelle accessibility through transient membrane gating represents a fundamental strategy by which eukaryotic cells could domesticate new endosymbiotic functions during early organellogenesis.

cell biology↗

bioRxiv2026

Balance between DNA repair, LINE1 suppression and lifespan in mice with SIRT6 Serine 10 mutations

Sirtuin 6 (SIRT6) is an important regulator of DNA repair, metabolism, chromatin maintenance and longevity. SIRT6 Serine 10 phosphorylation controls SIRT6 recruitment to the sites of DNA damage. To explore the effect of SIRT6 Serine 10 phosphorylation on lifespan, we generated two SIRT6 mutant mouse strains: phospho-null S10A and phosphomimetic S10E. The S10E mutant mice demonstrated enhanced DNA repair capacity, elevated LINE1 expression and reduced lifespan in male mice compared to the wild-type and S10A mice. This result suggests that SIRT6 S10E mutation enhances DNA repair capacity at the expense of reduced LINE1 silencing leading to shorter lifespan. While both SIRT6 functions in DNA repair and chromatin maintenance are important for longevity, our results suggest that when the balance between these functions is shifted, diminished of LINE1 control has a stronger impact on lifespan than enhanced DNA repair.

molecular biology↗

bioRxiv2026

Continuous mass photometry by single molecule trapping

The dynamic choreography of biomolecular interactions underpins the processes of life, but its direct observation remains challenging. Here, we introduce confined diffusion mass photometry, enabling hour-long, mass-resolved observation of individual biomolecules, their complexes and interactions with up to sub-kDa mass precision and ms temporal resolution. Our approach represents a quantitative time-resolved single-molecule measurement modality for studying complex biomolecular mechanisms in action.

biophysics↗

bioRxiv2025

Efagins evolved independently to target the enterococcal cell wall

Enterococci are major causes of multidrug-resistant infections, and antimicrobials with fundamentally new mechanisms of action are urgently needed. We identify a new class of antibacterial agents, termed efagins, which are chromosomally encoded, phage-related nanomachines that recognize cell wall carbohydrate receptors and inhibit subsets of E. faecalis, E. faecium and other enterococci selectively--a key reason they evaded prior detection. Five natural variants with distinct targeting profiles were identified - four related by sequence divergence, while one appears to have arisen through recombination of the targeting domain, likely from a phage donor. Operons encoding the corresponding carbohydrate receptors are highly variable, accounting for targeting specificity. The efagin targeting domain can be engineered to reprogram them toward alternative receptors, providing a pathway for filling critical coverage gaps. These findings advance efagins as new selective antibacterials with promise for addressing infection and spread of multidrug-resistant enterococcal infection.

microbiology↗

bioRxiv2025

Dual Membrane-spanning Anti-Sigma 2 Controls OMV biogenesis and Colonization Fitness in Bacteroides thetaiotaomicron

Bacteroides spp. are Gram-negative, gut commensals that shape the enteric landscape by producing outer membrane vesicles (OMVs) that degrade dietary fibers and traffic immunomodulatory biomolecules. Understanding the mechanism behind OMV biogenesis in Bacteroides spp. is necessary to determine their role in the gut. Recent studies showed that mutation of Dual Membrane-spanning Anti-sigma factor 1 increased OMV production in Bacteroides thetaiotaomicron (Bt) by regulating members of its downstream regulon. Additional members of the Dma family have been identified, but very little is known regarding their roles in Bt. Here, we investigate the role of Dual Membrane-spanning Anti-sigma factor 2 (Dma2) in controlling OMV biogenesis in Bt. We employ biochemical and proteomic analyses to show that mutation of dma2 increases OMV production in a manner that is dependent on the expression of its cognate sigma factor, das2. The precise mechanism by which dma2 increases OMV biogenesis remains elusive. However, transcriptome analyses revealed that{Delta} dma2 has decreased expression of select polysaccharide utilization loci (PULs) that primarily target host-associated glycans. Follow-up comparative proteomics showed that the PUL repertoire was most impacted in the OMV fraction. In vitro growth assessments showed that{Delta} dma2 exhibits delayed growth in the presence of select host-associated glycans. Colonization studies in mice revealed that{Delta} dma2 is outcompeted by the wild-type in the gut, which indicates that dma2 is a key determinant of colonization fitness in Bt. Altogether, these findings expand our knowledge of the Dma familys role in OMV biogenesis and demonstrates their importance in Bacteroides physiology. ImportanceDual membrane-spanning anti-sigma factors (Dma) are a novel class of regulatory system found solely amongst Bacteroidota. Previous studies have demonstrated the role of Dma1 in vesiculation, but the overall role of the Dma family in Bacteroides physiology remains poorly understood. Here, we demonstrate that Dma2 modulates vesiculation and regulates the expression of select polysaccharide utilization loci (PULs) that target host-associated glycans. In vivo studies revealed that Dma2 is an important fitness determinant when competing against kin bacteria. This work begins characterizing the multifaceted involvement of Dma2 in OMV biogenesis, PUL regulation, and colonization fitness.

microbiology↗

bioRxiv2025

In situ architecture of the endosymbiont Wolbachia pipientis

Hidden within host cells, the endosymbiont Wolbachia pipientis is the most prevalent bacterial infection in the animal kingdom. Scientific breakthroughs over the past century yielded fundamental mechanisms by which Wolbachia controls arthropod reproduction to shape dynamic ecological and evolutionary trajectories. However, the structure and spatial organization of symbiont machineries that underpin intracellular colonization and orchestrate maternal inheritance remain unknown. Here, we used cryo-electron tomography to directly image the nanoscale architecture of bacterial tools deployed for host manipulation and germline transmission. We discovered that Wolbachia assembles multiple structures at the host-endosymbiont interface including a filamentous ladder-like framework hypothesized to serve as a specialized motility mechanism that enables bacterial translocation to specific host cell compartments during embryogenesis and somatic tissue dissemination. In addition, we present the first in situ structure of the Rickettsiales vir homolog type IV secretion system (rvh T4SS). We provide evidence that the rvh T4SS nanomachine exhibits architectural similarities to the pED208-encoded T4SS apparatus including the biogenesis of rigid conjugative pili extending hundreds of nanometers beyond the bacterial cell surface. Coupled with integrative structural modeling, we demonstrate that in contrast to canonical T4SS architectures, the -proteobacterial T4SS outer membrane complex assembles a periplasmic baseplate structure predicted to comprise VirB9 oligomers complexed with cognate VirB10 subunits that form extended antennae projections surrounding the translocation channel pore. Collectively, these studies provide an unprecedented view into Wolbachia structural cell biology and unveil the molecular blueprints for architectural paradigms that reinforce ancient host-microbe symbioses.

microbiology↗

bioRxiv2025

M5 positive allosteric modulation alleviates parkinsonian motor deficits

Parkinsons disease is a neurodegenerative movement disorder which is characterized by cardinal motor symptoms of tremor at rest, rigidity, bradykineasia, and postural instability. Underlying these cardinal motor symptoms is thought to be death and dysfunction of nigrostriatal dopamine neurons, and the gold-standard treatment of Parkinsons disease is dopamine replacement therapy with the dopamine precursor L-DOPA. While efficacious, L-DOPA does not treat all motor symptoms and can have serious treatment-related side effects called L-DOPA induced dyskinesias, indicating an immense need for new targets to modulate dopaminergic function for anti-parkinsonian efficacy. One such potential target is the M5 muscarinic acetylcholine receptor, which has a unique expression profile where it is selectively expressed in midbrain dopaminergic neurons and their terminals in the striatum, and previous studies have indicated that M5 can modulate dopamine release and patterning of firing of dopamine neurons. Given this unique expression profile and function of M5, this receptor has an untested potential to modulate parkinsonian motor phenotypes. To test the potential for M5 to modulate Parkinsonian-like motor deficits and dyskinesia, we employed the unilateral 6-OHDA lesioned mouse model to create a hemi-parkinsonian state. Using multiple behavioral assays, including the cylinder test, forepaw adjusting steps assay, and in the Erasmus ladder, in conjunction with prototypical M5 pharmacological tool compounds, we investigated the ability of M5 to modulate parkinsonian motor deficits. Additionally, we tested the ability of M5 to modulate established L-DOPA induced dyskinesia or cause dyskinesia on its own. Overall, we found that M5 PAM alleviates forepaw asymmetry, bradykinesia, and spatial aspects of gait in the Erasmus ladder. Excitingly, M5 PAM does not cause robust dyskinesia, does not affect already established L-DOPA-induced dyskinesia, and does not affect L-DOPA motor efficacy. Taken together with previous findings, the current study suggests that M5 receptors are an exciting novel therapeutic strategy for ameliorating parkinsonian motor deficits even in late-stage models of severe PD without lessening L-DOPAs motor benefit and without affecting existing symptoms of L-DOPA-induced dyskinesia.

neuroscience↗

bioRxiv2025

A mathematical framework for human neutrophil state transitions inferred from single-cell RNA sequence data

Neutrophils, the most abundant immune cells in the human circulation, play a central role in the innate immune system. While neutrophil heterogeneity is a topic of increasing research interest, few efforts have been made to model the dynamics of neutrophil population subsets. We develop a mathematical model to describe the dynamics that characterizes the states and transitions involved in the maturation of human neutrophils. We use single-cell gene expression data to identify five clusters of healthy human neutrophils, and pseudo-time analysis to inform model structure. We find that precursor neutrophils transition into immature neutrophils, which then either transition to an interferon-responsive state or continue to mature through two further states. The key model parameters are the transition rates (the inverse of a transition rate is the mean waiting time in one state before transitioning to another). In this framework, the transition from the precursor to immature state (mean time less than an hour) is more rapid than subsequent transitions (mean times more than 12 hours). Approximately a quarter of neutrophils are estimated to follow the interferon-responsive path; the remainder continue along the standard maturation pathway. We use Bayesian inference to describe the variation, between individuals, in the fraction of cells within each cluster.

cell biology↗

bioRxiv2025

MotorBench: A Cryo-Electron Tomography Dataset of Bacterial Flagellar Motors for Testing Detection Algorithms

Understanding bacterial nanomachines like flagellar motors, which are crucial for pathogenic bacteria motility, is vital for microbiological and therapeutic research. Cryogenic electron tomography (cryo-ET) enables visualization of these structures within cells at near-native conditions. But manual identification remains challenging due to low contrast, limited resolution, and crowded in vivo environments. To address this, we introduce MotorBench, an expert-annotated dataset of bacterial flagellar motors that has been curated as part of a Kaggle competition BYU - Locating Bacterial Flagellar Motors 2025, engaging data scientists globally to create automated detection algorithms. MotorBench and its accompanying tools are intended to serve as a benchmark for evaluating and comparing future algorithms in automated cryo-ET analysis.

molecular biology↗

bioRxiv2024

Conditional Knockout of Striatal Gnal Produces Dystonia-like Motor Phenotypes

Loss-of-function mutations in GNAL have been linked to an adult-onset, isolated dystonia that is largely indistinguishable from idiopathic dystonia. GNAL encodes Golf, a heterotrimeric G-protein subunit with a defined molecular function to increase the production of the second messenger cAMP. Golf is abundant in the striatum, and is the only stimulatory G-protein in many cell types of the striatum. Due to the defined molecular signaling pathway and expression pattern of Golf, the clear genetic link to dystonia makes GNAL an exciting target to understand the pathological mechanisms of not only this genetic dystonia, but also the larger idiopathic disease. To better understand GNAL-linked dystonia, we generated a novel genetic mouse model that allows us to conditionally knock out Gnal in a site and time-specific manner. In the current study we used genetic or AAV based approaches to express Cre to knockout striatal Gnal in our novel Gnal fl/fl model. We then performed motor behavioral testing and ex vivo whole-cell patch clamp electrophysiology of striatal spiny projection neurons to interrogate how loss of Gnal leads to dystonia. Mice with conditional striatal knockout of Gnal show hindlimb clasping, other dystonia-like postures, less motor coordination, slowness, and torticollis as compared to age-matched controls. Furthermore, striatal spiny projection neurons show increased excitability in Gnal knockout animals. These exciting data are the first to report uninduced, overt dystonia in a mouse model of GNAL-linked dystonia, and directly correlate these with changes in spiny projection neuron electrophysiological properties. Our results show that adult loss of Gnal in the striatum leads to the development of dystonia, through homeostatic, paradoxical increases in spiny projection neuron excitability, and suggest that therapeutic strategies aimed at decreasing this hyperexcitable phenotype may provide symptomatic relief for patients with disease. One Sentence Summary: When Gnal is knocked out in the striatum of mice we observe overt behavioral symptoms and hyperexcitability in striatal spiny projection neurons.

neuroscience↗

bioRxiv2024

Open and closed conformations of a sub-80 kDa Chagas vaccine candidate defined by a cryo-EM led integrative approach

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a significant global public health concern. Despite its profound health impact in both endemic and non-endemic areas, no vaccine is available, and the existing therapies are outdated, producing severe side effects. The 80kDa prolyl oligopeptidase of Trypanosoma cruzi (TcPOP) has been recently identified as a leading candidate for Chagas vaccine development. We report the first three-dimensional structure of TcPOP in open and closed conformation, at a global resolution of 3.8 and 3.6 [A] respectively, determined using single-particle cryo-electron microscopy. Multiple conformations were observed and further characterized using plasmonic optical tweezers and hydrogen-deuterium exchange mass spectrometry. To assess the immunogenic potential of TcPOP, we immunized mice and evaluated both polyclonal and monoclonal responses against the TcPOP antigen and its homologues. The results revealed invasion blocking properties of anti-TcPOP polyclonal response via parasite lysis and its cross-reactivity versus closely-related POPs but not with human homologues. We were also able to produce and characterise three monoclonal antibodies, one of which showed neutralising properties and inhibition of parasite invasion via non-lytic mechanism. Collectively, our findings provide critical structural and functional insights necessary to understand the immunogenicity of TcPOP for future Chagas vaccine development and diagnostic applications.

biophysics↗

bioRxiv2023

ATOM-1: A Foundation Model for RNA Structure and Function Built on Chemical Mapping Data

RNA-based medicines and RNA-targeting drugs are emerging as promising new approaches for treating disease. Optimizing these therapeutics by naive experimental screening is a time-consuming and expensive process, while rational design requires an accurate understanding of the structure and function of RNA. To address this design challenge, we present ATOM-1, the first RNA foundation model trained on chemical mapping data, enabled by data collection strategies purposely developed for machine learning training. Using small probe neural networks on top of ATOM-1 embeddings, we demonstrate that this model has developed rich internal representations of RNA. Trained on limited amounts of additional data, these small networks achieve state-of-the-art accuracy on key RNA prediction tasks, suggesting that this approach can enable the design of therapies across the RNA landscape.

bioinformatics↗

bioRxiv2022

Structural remodeling of Coxiella burnetii during its biphasic developmental cycle revealedby cryo-electron tomography

Coxiella burnetii is an obligate zoonotic bacterium that targets macrophages to cause a disease known as Q fever. It has a biphasic developmental lifecycle where the extracellular and metabolically inactive small cell variant (SCV) transforms, under host acidic environment, into the vegetative large cell variant (LCV). However, the details about the morphological and structural changes that accompany this biphasic cycle are still lacking. Here, we used cryo-electron tomography to image the different cell variants of C. burnetii grown either under axenic conditions in different pH or purified directly from host cells revealing the major developmental, morphological and structural transitions. We show that SCVs are characterized by equidistant stacks of inner membrane that presumably allow a smooth transition to LCV, a transition coupled with the expression of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were associated with extracellular densities including a tubular structure possibly involved in host interaction or effector delivery. Also, SCVs and cells in the transition state contained spherical multilayered membrane structures of different sizes and locations suggesting that they are not related to a sporulation process as once assumed.

microbiology↗

bioRxiv2022

Highly Multiplexed Selection of RNA Aptamers Against a Small Molecule Library

Applications of synthetic biology spanning human health, industrial bioproduction, and ecosystem monitoring often require small molecule sensing capabilities, typically in the form of genetically encoded small molecule biosensors. Critical to the deployment of greater numbers of these systems are methods that support the rapid development of such biosensors against a broad range of small molecule targets. Here, we use a previously developed method for selection of RNA biosensors against unmodified small molecules (DRIVER) to perform a selection against a densely multiplexed mixture of small molecules, representative of those employed in high-throughput drug screening. Using a mixture of 5,120 target compounds randomly sampled from a large diversity drug screening library, we performed a 95-round selection and then analyzed the enriched RNA biosensor library using next generation sequencing (NGS). From our analysis, we identified RNA biosensors with at least 2-fold change in signal in the presence of at least 217 distinct target compounds with sensitivities down to 25 nM. Although many of these biosensors respond to multiple targets, clustering analysis indicated at least 150 different small-molecule sensing patterns. We also built a classifier that was able to predict whether the biosensors would respond to a new compound with an average precision of 0.82. Since the target compound library was designed to be representative of larger diversity compound libraries, we expect that the described approach can be used with similar compound libraries to identify aptamers against other small molecules with a similar success rate. The new RNA biosensors (or their component aptamers) described in this work can be further optimized and used in applications such as biosensing, gene control, or enzyme evolution. In addition, the data presented here provide an expanded compendium of new RNA aptamers compared to the 82 small molecule RNA aptamers published in the literature, allowing further bioinformatic analyses of the general classes of small molecules for which RNA aptamers can be found.

synthetic biology↗

bioRxiv2022

Dynamic structural adaptations enable the endobiotic predation of bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is an endobiotic microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here, we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometer-scale resolution. In addition to providing the first high-resolution images of predation in a native (hydrated, unstained) state, we also discover several surprising features of the process, including novel macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following replication and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/496000v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@35da74org.highwire.dtl.DTLVardef@19923f1org.highwire.dtl.DTLVardef@106a616org.highwire.dtl.DTLVardef@181f2fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗