Research

2024: Equine grass sickness is associated with major abnormalities in the ultrastructure of skeletal neuromuscular junctions

Bruce C McGorum , Tracey Davey , Miranda C M Dosi , John A Keen , Linda R Morrison , R Scott Pirie , Darren J Shaw , John B Harris

Equine grass sickness (EGS) is a frequently fatal multisystem neuropathy of equids. The aetiology is unknown; proposed causes include toxicoinfection with Clostridium botulinum and a mycotoxicosis. The effect of EGS on the organisation and structural integrity of the skeletal neuromuscular junction (NMJ), the target of botulinum neurotoxins (BoNTs), is unknown.

Results: A significantly higher percentage of EGS NMJs had abnormal morphology (EGS 72.2%, 95% CI 55.6-84.4; Controls 6.9%, 1.7-23.8; OR 35.1, 8.47-244.8; p < 0.001). EGS NMJs had a significantly lower mean volume fraction occupied by synaptic vesicles (SVs) (EGS 18.7%, 12.6-28.0; Controls 36.3%, 20.8-63.4; p = 0.024). EGS NMJs had evidence of accelerated SV exocytosis and SV depletion, accumulation of neurofilament-like material in terminal boutons and/or bouton degeneration. NMJs from the botulism horse had dense packing of SVs towards the presynaptic membrane active zone, consistent with BoNT intoxication, but had absence of the abnormalities identified in EGS NMJs.

Conclusions: EGS is associated with major changes in skeletal NMJ ultrastructure that are inconsistent with the effects of BoNTs. SV depletion may reflect increased exocytosis coupled with reduced repopulation of SVs via anterograde axonal transport and endocytosis, consistent with the action of an excitatory presynaptic toxin and/or neurotransmitter reuptake inhibitor. Skeletal NMJs represent a previously unrecognised target for the toxin that causes EGS.

Neuromuscular junctions from two equine grass sickness horses

Neuromuscular junctions from two equine grass sickness horses, showing varying synaptic vesicle (SV) densities across the respective terminals, with areas of high (solid star) and low (open star) SV density highlighted. There are occasional sites of SV fusion to terminal membranes (arrows) and variable SV sizes. Scale bar 500鈥塶m.

2022: Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment

Jennifer Ross, Zak McIver, Thomas Lambert, Cecilia Piergentili, Jasmine Emma Bird, Kelly J. Gallagher, Faye L. Cruickshank, Patrick James, Efrain Zaraz煤a-Arvizu4 Louise E. Horsfall, Kevin J. Waldron, Marcus D. Wilson, C. Logan Mackay, Arnaud Basl茅, David J. Clarke, Jon Marles-Wright.

Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo鈥揺lectron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/ deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Services used: TEM, Negative stain screening

Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFN尾 or IFN位1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

Catherine F. Hatton, Rachel A. Botting, Maria Emilia Due帽as, Iram J. Haq, Bernard Verdon, Benjamin J. Thompson, Jarmila Stremenova Spegarova, Florian Gothe, Emily Stephenson, Aaron I. Gardner, Sandra Murphy,Jonathan Scott,James P. Garnett,Sean Carrie, Jason Powell, C. M. Anjam Khan, Lei Huang, Rafiqul Hussain, Jonathan Coxhead, Tracey Davey, A. John Simpson, Muzlifah Haniffa, Sophie Hambleton, Malcolm Brodlie, Chris Ward,Matthias Trost, Gary Reynolds & Christopher J. A. Duncan

12, Article number: 7092 (2021)

Services used: TEM sample preparation,TEM

3D neuronal mitochondrial morphology in axons, dendrites, and somata of the aging mouse hippocampus

The brain's ability to process complex information relies on the constant supply of energy through aerobic respiration by mitochondria. Neurons contain three anatomically distinct compartments-the soma, dendrites, and projecting axons-which have different energetic and biochemical requirements, as well as different mitochondrial morphologies in cultured systems. In this study, we apply quantitative three-dimensional electron microscopy to map mitochondrial network morphology and complexity in the mouse brain. We examine somatic, dendritic, and axonal mitochondria in the dentate gyrus and cornu ammonis 1 (CA1) of the mouse hippocampus, two subregions with distinct principal cell types and functions. We also establish compartment-specific differences in mitochondrial morphology across these cell types between young and old mice, highlighting differences in age-related morphological recalibrations. Overall, these data define the nature of the neuronal mitochondrial network in the mouse hippocampus, providing a foundation to examine the role of mitochondrial morpho-function in the aging brain.

Julie Faitg, Clay Lacefield , Tracey Davey , Kathryn White , Ross Laws , Stylianos Kosmidis , Amy K Reeve , Eric R Kandel , Amy E Vincent , Martin Picard DOI:

Service used: Std TEM, SBF-SEM, Amira, Microscopy Image Browser ( MIB)

Quantitative 3D Mapping of the Human Skeletal Muscle Mitochondrial Network

Genetic and biochemical defects of mitochondrial function are a major cause of human disease, but their link to mitochondrial morphology in situ has not been defined. Here, we develop a quantitative three-dimensional approach to map mitochondrial network organization in human muscle at electron microscopyresolution. We establish morphological differences between human and mouse and among patients with mitochondrial DNA (mtDNA) diseases compared to healthy controls.

鈥�3D mitochondrial morphology is quantified by the mitochondrial complexity index (MCI)
鈥ouse mitochondria are larger and exhibit greater connectivity than human mitochondria
鈥on-adjacent mitochondria are connected by narrow mitochondrial nanotunnels
鈥ultivariate morphology signatures distinguish mitochondrial disease patients from healthy controls

Amy E. Vincent, Kathryn White, Tracey Davey, Jonathan Philips, R. Todd Ogden, Conor Lawless, Charlotte Warren, Matt G. Hall, Yi Shiau Ng, Gavin Falkous, Thomas Holden, David Deehan, Robert W. Taylor, Doug M. Turnbull, Martin Picard 2019

Service Used: Gatan 3View SBF-SEM

A guide to analysis and reconstruction of serial block face scanning electron microscopy data

Serial block face scanning electron microscopy (SBF鈥怱EM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers 鈥� how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D.

E. Cocks M. Taggart F.C. Rind K. White. Journal of Microscopy 2018

Service used: Gatan 3View. SBF Scanning Electron Microscopy. Amira. Microscopy Image Browser (MIB) Fiji. Blender

Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy

Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases.

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Sci Rep. 2016. Vincent AE, Ng YS, White K, Davey T, Mannella C, Falkous G, Feeney C, Schaefer AM, McFarland R, Gorman GS, Taylor RW, Turnbull DM, Picard M.

Service Used: Gatan 3View TEM

Reconstructed mitochondria

Trauma to Muller cells as the ILM is peeled from their attached end plates

A dissociated optic nerve fibre layer (DONFL) is a characteristic change noted in inner retinal morphology after internal limiting membrane (ILM) peeling. It is thought to be due to trauma to Muller cells as the ILM is peeled from their attached end plates.

Acta Ophthalmol. 2016 Steel DH, Dinah C, White K, Avery PJ.

Service used TEM , Stereology

Isolated proteins

There is increasing interest in the role of brain cholesterol in Alzheimer's disease and the contribution of cholesterol to the formation of amyloid plaques. Transmission EM can demonstrate the binding of soluble ~10 nm diameter cholesterol-PEG 600 micelles to amyloid-尾1-42 fibrils.

Micron 39:1192-6, 2008 - Harris JR.

Service used: TEM, Negative staining

Ultrastructural localization of Nova in the somata and dendrites of ventral horn spinal cord neurons

Front Neural Circuits 4:5, 2010 - Racca C, Gardiol A, Eom T, Ule J, Triller A, Darnell RB.

Service used: TEM, Pre-embedding immunogold

Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice

Diabetic glomerulopathy is characterised by mesangial expansion and thickening of the glomerular basement membrane. By applying stereological probes to digital images it is possible to quantify these changes in humans and experimental models.

Diabetes 57:2824-33, 2008 - Ku CH, White KE, Dei Cas A, Hayward A, Webster Z, Bilous R, Marshall S, Viberti GC, Gnudi L.

Service used: TEM, Stereology

Effect of neurotoxins on the neuromuscular junction

Examination of neuromuscular junctions using transmission EM can demonstrate how the injection of a neurotoxin into muscle results in a loss of synaptic vesicles from terminal boutons.

Histochem Cell Biol 132:395-404, 2009 - Rocha T, de Souza BM, Palma MS, da Cruz- H枚fling MA, Harris JB.

Service used: TEM, Standard epoxy resin embedding

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