Biology subjects

Montabana, E. A.

Publications and source records attributed to Montabana, E. A..

6 recordsLinked to original sources

bioRxiv2026

Characterization Standard for In-situ Cryo-electron Tomography

Standardized biological specimens are essential for optimizing cryoEM workflows and benchmarking instrument performance. While apoferritin fulfills this role for single-particle analysis, no equivalent exists for cryo-electron tomography. Ribosomes are frequently used but require large datasets due to C1 symmetry and structural heterogeneity, limiting rapid optimization and standardized comparison of workflows. Here, we present PP7 virus-like particles (VLPs) overexpressed in E. coli as a scalable in situ benchmark. VLPs have high orders of symmetry enabling rapid, high-resolution validation of tomographic pipelines from minimal datasets, while their distinct structural features across low to high resolutions provide a practical resolution metric.

cell biology↗

bioRxiv2026

Multiscale Physical Effects of CpG Methylation on DNA Mechanics, Nucleosome Wrapping, and Chromatin Condensates

DNA methylation at CpG dinucleotides is a central epigenetic modification linked to transcriptional repression and heterochromatin formation, yet its physical impact on chromatin organization remains incompletely understood. Here, we integrate single-molecule force spectroscopy, FRAP, and nanorheology to quantify how CpG methylation alters chromatin mechanics across scales. Optical tweezer-based measurements show that CpG methylation modestly increases DNA contour and persistence lengths, indicating a slightly extended and stiffer DNA helix. When assembled into nucleosomes, CpG methylation weakens both outer-and inner turn wrapping, reducing unwrapping forces and per-nucleosome unwrapping energy. On a higher-order assembly scale, although CpG methylated and unmethylated chromatin fibers display similar thresholds for salt-induced phase separation, methylated condensates exhibit markedly reduced internal mobility: FRAP reveals slower chromatin exchange, nanorheology shows increased viscoelasticity and longer relaxation times. Finally, CpG methylation enhances HP1-chromatin interactions, lowering the HP1 concentration required for condensation, despite no change in HP1 binding to naked DNA. Together, these results show that CpG methylation remodels chromatin mechanics by tuning DNA rigidity, nucleosome wrapping energetics, and condensate material properties, providing a multiscale physical framework for how DNA methylation contributes to heterochromatin.

biochemistry↗

bioRxiv2025

Cathepsin C-Catalyzed Ligation Generates Intralysosomal Amyloid Fibrils from Dipeptide Esters

The lysosome is a major catabolic organelle responsible for the breakdown of both intra- and extracellular substrates1,2. Lysosomal membrane damage mediated by pathologic amyloid fibrils is an area of recent focus3-6. The dipeptide ester LLOMe is typically employed to model lysosomal membrane damage7-11; however its mechanism of membranolysis was previously incompletely understood. Here, in vitro and cell-based analyses, and cryo-electron microscopy and tomography studies reveal LLOMe-derived oligopeptides generated by the lysosomal protease Cathepsin C assemble into cross-{beta}-sheet amyloid fibrils within the lysosome. Additionally, we report lysosome membrane damage triggers the broadly nonspecific dipeptidyl ligase activity of Cathepsin C, facilitating the tagging of proximal proteins within the damaged lysosome lumen with a click chemistry handle: to our knowledge, the first reported localized proximity labeling approach exploiting a fully endogenous, non-engineered enzyme. While Cathepsin C ligase activity has been demonstrated in vitro12,13, our observations of dipeptidyl ligation onto proximal proteins in cells suggests an unexplored role of Cathepsin C in lysosomal biology and broadly exemplifies how other endogenous enzymes might be similarly exploited for proximity labeling. Altogether our results unveil two mechanisms by which dipeptide esters perturb lysosomal homeostasis and provide a roadmap for their utilization toward targeted studies of the lysosome.

biochemistry↗

bioRxiv2025

AreTomoLive: Automated reconstruction of comprehensively-corrected and denoised cryo-electron tomograms in real-time and at high throughput

A high throughput processing pipeline that performs comprehensive corrections is needed to realize the full potential of cryo-electron tomography and subtomogram averaging. The fields fragmented software landscape remains a significant hurdle to this end. Here we present AreTomoLive, an automated real-time pipeline composed of two GPU-accelerated packages. The first, AreTomo3, streamlines tomographic alignment and reconstruction, with new features to fully account for sample geometry, locally correct the contrast transfer function, and curate data for downstream tasks. The second package, DenoisET, is a new implementation of the machine learning algorithm Noise2Noise and runs in parallel with AreTomo3 to perform contrast enhancement. To reduce barriers to routine use, AreTomoLive prioritizes automation: AreTomo3 autonomously pauses and reactivates processing depending on the status of data collection, while DenoisET algorithmically determines when to transition from training to inference. AreTomoLive endeavors to advance cryoET for in situ structural analysis with its comprehensive corrections and full automation.

cell biology↗

bioRxiv2022

Candidatus Nealsonbacteria (OD1) in a methanogenic benzene-degrading enrichment culture is likely an ectosymbiotic biomass recycler

The Candidate Phyla Radiation (CPR) is a very large group of bacteria with no pure culture representatives, first discovered by metagenomic analyses. Within the CPR, candidate phylum Parcubacteria (previously referred to as OD1) within the candidate superphylum Patescibacteria is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the Candidate clade Nealsonbacteria. Because of its persistence over many years, we hypothesized that Ca. Nealsonbacteria DGGOD1a must serve an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10 fold) increase in the absolute abundance of Ca. Nealsonbacteria DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate Ca. Nealsonbacteria in biomass recycling. Fluorescent in situ hybridization and cryogenic transmission electron microscope images revealed that Ca. Nealsonbacteria DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other Ca. Nealsonbacteria found in anoxic environments.

microbiology↗

bioRxiv2020

Lattice micropatterning of electron microscopy grids for improved cellular cryo-electron tomography throughput

Cryo-electron tomography is the highest resolution tool available for structural analysis of macromolecular complexes within their native cellular environment. At present, data acquisition suffers from low throughput, in part due to the low probability of positioning a cell such that the subcellular structure of interest is on a region of the electron microscopy (EM) grid that is suitable for imaging. Here, we photo-micropatterned EM grids to optimally position endothelial cells so as to enable high-throughput imaging of cell-cell contacts. Lattice micropatterned grids increased the average distance between intercellular contacts and the thicker cell nuclei such that the regions of interest were sufficiently thin for direct imaging. We observed a diverse array of membranous and cytoskeletal structures at intercellular contacts, demonstrating the utility of this technique in enhancing the rate of data acquisition for cellular cryo-electron tomography studies.

bioengineering↗