Biology subjects

Grotjahn, D.

Publications and source records attributed to Grotjahn, D..

12 recordsLinked to original sources

bioRxiv2026

Imaging large fields-of-view at high resolution in cryo-ET with square beam montaging

Visualizing macromolecules within their native cellular context by cryo-electron tomography (cryo-ET) is fundamentally limited by the trade-off between field of view and resolution: Capturing high-resolution information about biomolecules requires high magnification, which restricts the field of view and obscures the cellular context in which those biomolecules function. Collecting montage data by tiling the electron beam over the region of interest offers one solution, although traditional round electron beams cause excessive radiation damage across overlapping regions. We previously made electron beams square in shape, enabling montage collection with minimal overlap and thereby reducing excessive exposure and loss of high-resolution information. Here, we create a pipeline for collecting and processing montage cryo-ET data with square electron beams. We show that square beam montages retain high-resolution information by reconstructing virus-like particles to 3.5 [A] resolution using sub-tomogram averaging, and apply the workflow to imaging a glial cell and malaria parasite lamellae over fields of view up to 65 m2. We also provide a comprehensive protocol to make square beams accessible to the community.

molecular biology↗

bioRxiv2026

ATAD3A structurally links mtDNA replication and mitochondrial fission

Mitochondrial function depends on the maintenance of its genome, and disruptions in copy number and distribution are hallmarks of mitochondrial disorders. Mitochondrial DNA (mtDNA) replication is spatially and temporally linked to mitochondrial division (i.e., fission). However, the signal that coordinates these two events, which are physically separated by the barrier of two mitochondrial membranes, remains unknown. To gain insight into this coordination, we employed correlative cryo-electron tomography (cryo-ET) to analyze the microenvironment surrounding replicating nucleoids. Mitochondrial regions containing replicating mtDNA exhibit a unique membrane architecture defined by the presence of clustered, membrane-spanning tethers that traverse the inner membrane space. Using a combination of superresolution microscopy and genetically encoded cryo-ET tagging technology, we identify these tethers as the AAA+ ATPase ATAD3A. We further show that ATAD3A knockdown reduces recruitment of the mitochondrial fission machinery, whereas overexpression promotes its recruitment and subsequent fission. Our work suggests that ATAD3A forms nanoscale linkages that coordinate these two distinct processes, revealing a new structural paradigm for organellar communication across distinct membrane-defined environments. HighlightsO_LIReplicating mitochondrial DNA (mtDNA) nucleoids are surrounded by a distinct membrane microenvironment. C_LIO_LIATAD3A forms membrane-spanning tethers enriched at replicating mtDNA sites. C_LIO_LIATAD3A enrichment is necessary and sufficient to recruit mitochondrial fission machinery and induce fission at mtDNA replication sites. C_LIO_LIATAD3A structurally couples mtDNA replication state to mitochondrial fission across distinct organellar subcompartments. C_LI

Cell Biology↗

bioRxiv2026

DM: a simple solution to suppress air-water interface interactions in cryo-EM

The air-water interface (AWI) remains the primary barrier to routine high-resolution cryo-EM structure determination, driving protein adsorption, structural denaturation, and restricted particle orientations during vitrification. Here, we describe a simple and broadly applicable strategy to mitigate these effects using the mild non-ionic detergent n-decyl-{beta}-D-maltopyranoside (DM). Addition of DM at low millimolar concentrations immediately prior to vitrification consistently suppresses AWI-driven artifacts, resulting in improved angular sampling, reduced structural damage, and enhanced reconstruction quality across diverse macromolecular systems. Using this approach, we obtained a high-resolution reconstruction of the 65 kDa Nucleophosmin 1 pentamer, a target previously limited by severe preferred orientation issues. We further show that DM promotes isotropic particle distributions for high-resolution reconstruction of hemagglutinin, transthyretin, as well as suppressing denaturation of aldolase while stabilizing its C-terminus. Our results indicate that DM effectively passivates deleterious air-water interface interactions without compromising particle integrity. These results establish DM as an effective additive for improving the robustness of single-particle cryo-EM sample preparation. O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/716008v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@108a6edorg.highwire.dtl.DTLVardef@10728b4org.highwire.dtl.DTLVardef@1014b2eorg.highwire.dtl.DTLVardef@1eed745_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Drug Repurposing Screen Identifies an HRI Activating Compound that Promotes Adaptive Mitochondrial Remodeling in MFN2-deficient Cells

Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth Type 2A (CMT2A). These mutations disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR) - the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2-deficient cells. These results demonstrate the potential for pharmacologic HRI activation as a viable strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.

cell biology↗

bioRxiv2025

Surface morphometrics reveals local membrane thickness variation in organellar subcompartments

Lipid bilayers form the basis of organellar architecture, structure, and compartmentalization in the cell. Decades of biophysical, biochemical, and imaging studies on purified or in vitro reconstituted liposomes have shown that variations in lipid composition influence the physical properties of membranes, such as thickness and curvature. However, similar studies characterizing these membrane properties within the native cellular context have remained technically challenging. Recent advancements in cellular cryo-electron tomography (cryo-ET) imaging enable high-resolution, three-dimensional views of native organellar membrane architecture preserved in near-native conditions. We previously developed a Surface Morphometrics pipeline that generates surface mesh reconstructions to model and quantify cellular membrane ultrastructure from cryo-ET data. Here, we expand this pipeline to measure the distance between the phospholipid head groups (PHG) of the membrane bilayer as a readout of membrane thickness. Using this approach, we demonstrate thickness variations both within and between distinct organellar membranes. We also demonstrate that membrane thickness positively correlates with other features, such as membrane curvedness. Further, we show that subcompartments of the mitochondrial inner membrane exhibit varying membrane thicknesses that are independent of network morphology (i.e., fragmented versus elongated networks). Finally, we demonstrate that large membrane-associated macromolecular complexes exhibit distinct density profiles that correlate with local variations in membrane thickness. Overall, our updated Surface Morphometrics pipeline provides a framework for investigating how changes in membrane composition in various cellular and disease contexts affect organelle ultrastructure and function.

biochemistry↗

bioRxiv2024

scLong: A Billion-Parameter Foundation Model for Capturing Long-Range Gene Context in Single-Cell Transcriptomics

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of cellular heterogeneity by providing gene expression data at single-cell resolution, uncovering insights into rare cell populations, cell-cell interactions, and gene regulation. Foundation models pretrained on large-scale scRNA-seq datasets have shown great promise in analyzing such data, but existing approaches are often limited to modeling a small subset of highly expressed genes and lack the integration of external genespecific knowledge. To address these limitations, we present sc-Long, a billion-parameter foundation model pretrained on 48 million cells. sc-Long performs self-attention across the entire set of 28,000 genes in the human genome. This enables the model to capture long-range dependencies between all genes, including lowly expressed ones, which often play critical roles in cellular processes but are typically excluded by existing foundation models. Additionally, sc-Long integrates gene knowledge from the Gene Ontology using a graph convolutional network, enriching its contextual understanding of gene functions and relationships. In extensive evaluations, sc-Long surpasses both stateof-the-art scRNA-seq foundation models and task-specific models across diverse tasks, including predicting transcriptional responses to genetic and chemical perturbations, forecasting cancer drug responses, and inferring gene regulatory networks.

bioinformatics↗

bioRxiv2024

ProteinAligner: A Multi-modal Pretraining Framework for Protein Foundation Models

Protein foundation models, particularly protein language models, have demonstrated strong success in learning meaningful representations of proteins using transformer architectures pretrained on large-scale protein datasets with self-supervised learning. These representations have been highly effective for downstream tasks such as predicting protein functions and properties. However, most current protein foundation models focus on pretraining with amino acid sequences, often neglecting additional modalities like protein structures and related literature, both of which provide valuable insights. To address this gap, we propose a multi-modal pretraining approach that integrates three key modalities - protein sequences, structures, and literature text. In our framework, the protein sequence modality serves as the anchor, with the other two modalities aligned to it, enhancing the models capacity to capture more comprehensive protein information. ProteinAligner out-performed state-of-the-art protein foundation models in predicting protein functions and properties across diverse down-stream tasks.

bioinformatics↗

bioRxiv2024

Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2 kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that pharmacologic activation of the ISR reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

cell biology↗

bioRxiv2023

Secreted antigen A peptidoglycan hydrolase is essential for Enterococcus faecium cell separation and priming of immune checkpoint inhibitor cancer therapy

Enterococcus faecium is a microbiota species in humans that can modulate host immunity1, but has also acquired antibiotic resistance and is a major cause of hospital-associated infections2. Notably, diverse strains of E. faecium produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity3-5 and immune checkpoint inhibitor antitumor activity6. However, the functions of SagA in E. faecium were unknown. Here we report that deletion of sagA impaired E. faecium growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, {Delta}sagA showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, plasmid-based expression of SagA, but not its catalytically-inactive mutant, restored {Delta}sagA growth, production of active muropeptides and NOD2 activation. SagA is therefore essential for E. faecium growth, stress resistance and activation of host immunity.

microbiology↗

bioRxiv2023

Overcoming Resolution Attenuation During Tilted Cryo-EM Data Collection

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60{degrees} are virtually indistinguishable. These strategies allowed the reconstruction to 3 [A] resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

biophysics↗

bioRxiv2022

PERK Signaling Promotes Mitochondrial Elongation By Remodeling Membrane Phosphatidic Acid

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are linked in the onset and pathogenesis of numerous diseases. This has led to considerable interest in defining the mechanisms responsible for regulating mitochondria during ER stress. The PERK signaling arm of the unfolded protein response (UPR) has emerged as a prominent ER stress-responsive signaling pathway that regulates diverse aspects of mitochondrial biology. Here, we show that PERK activity also promotes adaptive remodeling of mitochondrial membrane phosphatidic acid (PA) to induce protective mitochondrial elongation during ER stress. We find that PERK activity is required for ER stress-dependent increases in both cellular PA and YME1L-dependent degradation of the intramitochondrial PA transporter PRELID1. Together, these processes lead to the accumulation of PA on the outer mitochondrial membrane where it induces mitochondrial elongation by inhibiting mitochondrial fission. Our results establish a new role for PERK in the adaptive remodeling of mitochondrial phospholipids and demonstrate that PERK-dependent PA regulation functions to adapt organellar shape in response to ER stress.

cell biology↗

bioRxiv2020

A guided approach for subtomogram averaging of challenging macromolecular assemblies

Cryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

molecular biology↗