Biology subjects

Bobe, D.

Publications and source records attributed to Bobe, D..

10 recordsLinked to original sources

bioRxiv2026

A robust workflow for 3D imaging of human mitochondria using cryo-electron tomography

Mitochondria are dynamic signaling organelles that transduce metabolic and biochemical cues to facilitate cellular adaptation. Their complex structure and dynamics are essential for integrating metabolic pathways, responding to stressors, and communicating inter- and intra-cellular signals. While optimal mitochondrial activity is frequently linked to cellular and organismal health--influencing processes ranging from metabolism and regulated cell death to differentiation and growth--the mechanistic links between mitochondrial dysfunction and cellular defects leading to human disease remain incompletely understood. Understanding how mitochondrial shape and function are linked is crucial for deciphering the regulatory mechanisms of cell survival and fate. Here, we present a molecular resolution cryo-electron tomography (cryo-ET) imaging and image analysis platform to investigate the structure of isolated human mitochondria under different conditions. We describe optimized protocols for isolating mitochondria from human cells, vitrifying these samples with high-pressure freezing (HPF) using the waffle method, cryo-focused ion beam (cryo-FIB) milling to generate thin sections (lamellae), and imaging with cryo-transmission electron microscopy (cryo-TEM). This is complemented by a robust downstream processing pipeline for tilt-series alignment, tomogram reconstruction, and three-dimensional (3D) segmentation of tomograms using the latest state-of-the-art algorithms. With some variations, this versatile workflow is adaptable to other subcellular compartments for structural studies in isolation or within intact cells. Furthermore, our protocols provide a critical foundation for investigating the in-situ structure of protein machineries that govern key cellular processes.

biophysics↗

bioRxiv2026

Eg5 activity and density-driven bundling organize the human metaphase mitotic spindle independently of spindle bipolarity

The mitotic spindle segregates chromosomes through the coordinated actions of microtubules and molecular motors. Classic models propose that microtubules nucleate at spindle poles and grow inward to capture chromosomes; however, recent structural studies show that spindles contain short microtubules that do not span the distance between poles and chromosomes. It is unclear how these short microtubules assemble a bipolar spindle. Using cryo-electron tomography to map microtubule polarity in human metaphase spindles, we find that microtubules form locally antiparallel bundles with consistent 8 nm wall-to-wall spacing. We utilized motor perturbations and centriole depletion, which generated motor-active monopolar spindles, to reveal that the kinesin-5 motor Eg5 organizes local antiparallel overlap independently of spindle bipolarity. We found that bundles are organized by density-driven steric interactions rather than motor-mediated crosslinking. These findings support a self-organized, bottom-up model in which local microtubule-motor interactions within dense bundles generate forces that build the bipolar spindle, challenging pole-centric models. KEY FINDINGSO_LIMicrotubules in metaphase spindles organize in locally antiparallel bundles with consistent 8 nm wall-to-wall spacing. C_LIO_LIThe Eg5 motor generates antiparallel microtubule overlap independently of spindle bipolarity. C_LIO_LIMicrotubule spacing scales with density through steric interactions, not direct motor crosslinking. C_LIO_LIDynein regulates microtubule density to control bundle architecture; balanced Eg5-dynein density regulation maintains spindle bipolarity. C_LI

biophysics↗

bioRxiv2025

Understanding the infection mechanism of {Phi}8 using infection method in Pseudomonas syringae pv. Phaseolicola strain: In-situ CryoET analysis

The Cystoviridae family strain {Phi}8 is an ideal model for studying the virus-host interactions and viral assembly mechanisms. In this study, we explored {Phi}8 infection kinetics in P. syringae pv. phaseolicola host cells using a combination of host lysis assays, transmission electron microscopy (TEM), cryo-focused ion beam (Cryo-FIB) milling, and in-situ cryo-electron tomography (Cryo-ET). Purified {Phi}8 was infected with host cells and observed at different time points. The host-pathogen infection was detected using TEM studies. The infected sample underwent Cryo-FIB milling. Our findings indicated that the infected host cell membrane changes its shape during infection. We were able to visualize the various stages of {Phi}8 assembly using subtomogram averaging. These stages included the unpackaged procapsid (PC) having a size of [~]41 nm and the double-stranded RNA (dsRNA)-packaged procapsid having a size of [~]54 nm. The packaged PC represented the most prevalent intermediate stages during viral assembly. Notably, the majority of complete virions isolated from the host cell were found outside the host cell, revealing the extracellular predominance of fully constructed particles. These findings emphasize the importance of stable intermediate stages in viral assembly and maturation of {Phi}8. This work advances our understanding of viral assembly and complex host-cell interactions. HighlightsO_LISuccessfully isolated and infected Pseudomonas syringae pv. phaseolicola (Pph) host cells with {Phi}8. C_LIO_LILysis assay performed to quantify host lysis to evaluate viral infection efficiency. C_LIO_LICryo-focused ion beam milling enabled precise preparation of cellular cross-sections for structural analysis. This allows identification of key stages of the {Phi}8 life cycle during the infection cycle. C_LIO_LIThe majority of complete virions were localized extracellularly, emphasizing the role of extracellular stages in the {Phi}8 lifecycle. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/678632v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@6ba760org.highwire.dtl.DTLVardef@1b99d2dorg.highwire.dtl.DTLVardef@fdfe72org.highwire.dtl.DTLVardef@14c077e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Overcoming air-water interface induced artifacts in Cryo-EM with protein nanocrates

Contact with the air-water interface can bias the orientation of macromolecules during cryo-EM sample preparation, leading to uneven sample distribution, preferred orientation, and damage to the molecules of interest. To prevent this, we describe a method to encapsulate target proteins within highly hydrophilic, structurally homogeneous, and stable protein shells, which we refer to as "nanocrates" for this purpose. Here, we describe packaging, data acquisition, and reconstruction of three proof-of-principle examples, each illuminating a different aspect of the method: apoferritin (ApoF, demonstrating high-resolution), thyroglobulin (Tg, solving a known preferred orientation problem), and 7,8-dihydroneopterin aldolase (DHNA, a structure previously uncharacterized by cryo-EM).

bioengineering↗

bioRxiv2024

Identification and cryoEM structure determination of Escherichia phage YDC107 tail found in a bacteria-contaminated buffer

Cryo-electron microscopy data analysis can yield multiple structures from a single heterogeneous dataset. Here, we show a workflow we used for the identification of a contaminant from a cryoEM grid without prior knowledge of protein sequence. We determined the tail structure of Escherichia phage YDC107 from only several thousand particles. The workflow combines high-resolution single-particle data processing with de novo model determination using ML-based methods. Structural analysis revealed that the central part of the phage tail has a C6 symmetry, however the overall symmetry of each segment is C3 due to dimerization of a flexible domain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/627647v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1444ae3org.highwire.dtl.DTLVardef@907ec1org.highwire.dtl.DTLVardef@71eebdorg.highwire.dtl.DTLVardef@1f0e6a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Multi-species cryoEM calibration and workflow verification standard

Cryogenic electron microscopy (cryoEM) is a rapidly growing structural biology modality that has been successful in revealing molecular details of biological systems. However, unlike established biophysical and analytical techniques with calibration standards, cryoEM has lacked comprehensive biological test samples. We introduce a cryoEM calibration sample that is a mixture of compatible macromolecules that can be used not only for resolution optimization but also provides multiple reference points for evaluating instrument performance, data quality, and image processing workflows in a single experiment. This combined test specimen provides researchers a reference point for validating their cryoEM pipeline, benchmarking their methodologies, and testing new algorithms.

biochemistry↗

bioRxiv2024

Cryo-ET reveals the in situ architecture of the polar tube invasion apparatus from microsporidian parasites

Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis. Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ, laying the foundation for understanding infection mechanisms.

microbiology↗

bioRxiv2024

Environmental differences impact Acinetobacter baumannii phage isolation and infectivity

With the global rise of antimicrobial resistance, phage therapy is increasingly re-gaining traction as a strategy to treat bacterial infections. For phage therapy to be successful however, we first need to isolate appropriate candidate phages for both clinical and experimental research. Acinetobacter baumannii is an opportunistic pathogen known for its ability to rapidly evolve resistance to antibiotics, making it a prime target for phage therapy. Yet phage isolation is often hampered by A. baumanniis ability to rapidly switch between capsular states. Here, we report the discovery and structural characterisation of a novel lytic phage, Mystique. This phage was initially isolated against the wild-type AB5075: a commonly used clinical model strain against which no phage has previously been readily available for the capsulated form. When screening Mystique on 103 highly diverse isolates of A. baumannii, we found that it has a broad host range, being able to infect 85.4% of all tested strains when tested on bacterial lawns - a host range which expanded to 91.3% when tested in liquid culture. This variation between solid and liquid environments on phage infectivity was also observed for several other phages in our collection that were assumed unable to infect AB5075, and capsule negative mutants that initially seemed completely resistant to Mystique proved susceptible when assayed in liquid. Overall, through the discovery of a novel phage we demonstrate how environmental differences can drastically impact phage infectivity with important consequences for phage isolation and characterisation efforts. Author summaryBacterial infections caused by Acinetobacter baumannii are a major global health concern due to high antibiotic resistance, earning it a critical priority pathogen ranking by the WHO. Phage therapy is resurging as a treatment option, with some success against A. baumannii. However, the wild-type clinical model strain used to assess new therapies lacks an available phage, and isolating phages for A. baumannii is challenging due to its complex capsule. Here, we report the discovery of a novel lytic phage, Mystique, which exhibits a broad host range, infecting 94 out of 103 tested A. baumannii strains. We conducted genomic sequencing and structural analysis to fully characterise Mystique. Additionally, we found that the testing environment significantly impacts results; some phages that do not form plaques on bacterial lawns can still infect and amplify in liquid cultures of the same strain. Moreover, mutants resistant to Mystique based on plaque assays were susceptible in liquid culture assays. This work underscores the necessity of a multifaceted approach for phage isolation and characterisation, as traditional phage assays may not be sufficient for studying bacteria-phage dynamics in certain bacteria such as A. baumannii.

microbiology↗

bioRxiv2020

Time-resolved cryoEM using Spotiton

We present an approach for preparing cryoEM grids to study short-lived molecular states. Using piezo electric dispensing, two independent streams of ~50 pL sample drops are deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing reaction stops when the grid is vitrified in liquid ethane, on the order of ~100 ms later. We demonstrate the utility of this approach for four biological systems where short-lived states are of high interest.

biophysics↗

bioRxiv2018

Benchmarking cryo-EM single particle analysis workflow

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.

molecular biology↗