Biology subjects

Hanssen, E.

Publications and source records attributed to Hanssen, E..

15 recordsLinked to original sources

bioRxiv2026

Whole cryo-FIB lamella acquisition at high magnification with rectangular beam montage cryo-electron tomography

The visualisation of large biological samples in cryo-electron tomography requires both few-nanometre resolution and a wide, ideally >10 micrometrre, field of view. For a single image, increasing magnification trades increased resolution for a smaller field of view. Montaging, where high-magnification images are stitched together to increase field of view, addresses the trade-off but is dose-inefficient with round beams that are standard in transmission electron microscopy (TEM), since this illumination shape does not tessellate. Our workflow greatly simplifies montaging schemes for dose-sensitive specimens, allowing large montages of any shape - from individual cells or other features of interest up to whole lamellae - to be collected at high resolution without compromising electron dose efficiency. We demonstrate the workflow by acquiring montage tomograms of whole yeast (Saccharomyces cerevisiae) cells in cryo-FIB lamellae and of human neurons.

biophysics↗

bioRxiv2025

The trimeric structures of the extracellular domains of FAM171A1 and FAM171A2 neuronal proteins belong to a novel structural superfamily

Cell surface molecules play fundamental roles in cell-cell communication, attraction, or repulsion, and when expressed in neurons they are often implicated in neurological disorders. FAM171 is a family of three type-I transmembrane domain cell surface proteins (FAM171A1, FAM171A2, and FAM171B) expressed in several human tissues and especially enriched in the brain. Recent findings suggest that FAM171A1 transduces signals between the cell surface and the cytoskeleton. Genetic evidence links FAM171A1 to multiple cancers and FAM171A2 to neurodegenerative diseases, including Alzheimers and Parkinsons diseases. Despite multiple connections with severe human diseases, no information is currently available on their monomeric structure or oligomerization. Here we show that, structurally, the monomeric ectodomains of human FAM171A1 and FAM171A2 have a new architecture with a novel combination of two domains. Furthermore, their ectodomains oligomerize to form an equilateral trimer. In addition, the ectodomain of FAM171A1 has the propensity to form larger trimer-trimer assemblies at high concentrations. Together, these results provide novel insights into the structure and oligomerization of the extracellular domain of FAM171A1 and FAM171A2, suggesting important roles in ligand binding and signaling.

neuroscience↗

bioRxiv2025

Electron tomography reveals mitochondrial network and cristae remodelling during cell differentiation in the human placenta

Mitochondria adapt their structure through fusion and fission, yet how their morphology and cristae architecture alter as cells differentiate remains unclear. The human placenta is an ideal model for studying mitochondrial diversity within a single tissue. As epithelial trophoblast cells of the placenta differentiate, their accompanying mitochondria morphologically and functionally transform. We used array tomography to characterise mitochondrial volume and network complexity. Cryo-electron tomography revealed two distinct subpopulations of mitochondria within progenitor cytotrophoblasts, and a singular, homogeneous population in the differentiated syncytiotrophoblast. We showcased the 3D topology of individual cristae through a standardised metric of "curvedness". Proteomic analysis of isolated mitochondria identified reduced dynamics proteins and cristae organisation complexes, accompanied by subunit variations within the electron transport chain, ATP synthase, and altered supercomplex abundance. This study highlights the advantages of combining multimodal imaging with mechanistic insights, to elucidate the process of mitochondrial morphology and cristae architecture remodelling as cells differentiate.

cell biology↗

bioRxiv2025

Near-atomic structure of the PorKN rings, disulfide bonded to PorG and bound to Attachment Complexes, provide mechanistic insights into the type IX secretion system

The Type IX Secretion System exports proteins across the outer membrane (OM) of bacteria in the Bacteroidota phylum, however, the mechanistic details remain unknown. Here, we present a [~]3.5[A] cryo-EM structure of the periplasmic rings comprising 32-33 subunits each of PorK and PorN. Additionally, we show the presence of a critical disulfide bond between PorK and the PorG OM protein that is essential for protein secretion and demonstrate that the Attachment Complexes bind to and are localized above the PorKN rings. Overall, each ring resembles a cogwheel with PorN forming cog-like projections on the periplasmic side and the flat surface of PorK orienting towards the OM. Given these results, we propose that the PorLM motor drives the rotation of the PorKN cogwheel together with PorG and associated Attachment Complexes, potentially providing the energy to complete protein secretion and the coordinated cell surface attachment of the secreted cargo.

microbiology↗

bioRxiv2024

Opposing roles of resident and infiltrating immune cells in the defence against Legionella longbeachae via IL-18R/IFN-γ/ROS axis

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires pneumonia, is poorly understood. Here we investigated the specific roles of tissue-resident alveolar macrophages (AM) and infiltrating phagocytes during infection with this pathogen. AM were the predominant cell type that phagocytosed bacteria o day after infection. Three and five days after infection, AM numbers were greatly reduced while there was an influx of neutrophils and later monocyte-derived cells (MC) into lung tissue. AM carried greater numbers of viable L. longbeachae than neutrophils and MC, which correlated with a higher capacity of L. longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection whereas neutrophils and MC were required for efficient bacterial clearance. IL-18 was important for IFN-{gamma} production by IL-18R+ NK cells and T cells which, in turn, stimulated ROS-mediated bactericidal activity in neutrophils resulting in restriction of L. longbeachae infection. Ciliated epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L. longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L. longbeachae infection that may be manipulated to improve protective responses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/578217v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@107c421org.highwire.dtl.DTLVardef@dd76f5org.highwire.dtl.DTLVardef@1acf746org.highwire.dtl.DTLVardef@9e2c17_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAM serve as a replicative niche and promote L. longbeachae infection. C_LIO_LIInfiltrating neutrophils and MC kill L. longbeachae. C_LIO_LIIFN-{gamma} from IL-18R+ NK and T cells stimulates ROS and bacterial clearance. C_LI

immunology↗

bioRxiv2023

Vgamma9Vdelta2 T cells recognize butyrophilin 2A1 and 3A1 heteromers

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity, however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell receptor ({gamma}{delta}TCR) in complex with BTN member 2A1 (BTN2A1) revealed that BTN2A1 engages the side of the {gamma}{delta}TCR, leaving the apical TCR surface bioavailable. We reveal that BTN3A1 is a second {gamma}{delta}TCR ligand, that co-engages {gamma}{delta}TCR via binding to this accessible apical surface. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to engage {gamma}{delta}TCR; indeed, either forced separation or locking together of BTN2A1 and BTN3A1 resulted in enhanced or abrogated {gamma}{delta}TCR interaction, respectively. Our findings reveal a new paradigm in immune activation, whereby {gamma}{delta}TCRs recognize dual epitopes on BTN2A1 and BTN3A1 complexes.

immunology↗

bioRxiv2023

Fitting a square beam in a square camera: novel condensor apertures for low-dose transmission electron microscopy

In transmission electron microscopy (TEM) cameras are square or rectangular but beams are round. With a beam size chosen to fill the camera at a given image magnification, the circular lobes of the beam will extend beyond the cameras field of view and irradiate areas that are not acquired on the camera, damaging and precluding them from future acquisitions if the sample is beam sensitive. In this paper we present development of condenser aperture plates for TEM that have square and rectangular apertures which improve the efficiency of sample area usage by 44% or greater in low dose TEM applications. We demonstrate that the use of these apertures is compatible with high-resolution cryogenic (cryo) TEM by reconstructing sub 2 [A] apo-ferritin models from a datasets recorded with both square and rectangular apertures. Moreover the design of our aperture plates should improve the flexibility of 2 condenser systems for cryo-TEM acquisitions with multiple shots per hole by tailored matching of beam sizes to camera sizes at each magnification.

biophysics↗

bioRxiv2022

Structures of the interleukin 11 signalling complex reveal dynamics of gp130 extracellular domains and the inhibitory mechanism of a cytokine variant

Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in numerous autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors is lacking. Here we present cryo-EM and crystal structures of the IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family {beta}-receptor, gp130. We show that the membrane-proximal domains of gp130 are dynamic and do not participate in complex assembly. We demonstrate that the cytokine mutant IL-11 Mutein competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibitory activity by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.

cell biology↗

bioRxiv2022

Effects of altered cellular ultrastructure on energy metabolism in diabetic cardiomyopathy - an in-silico study

Diabetic cardiomyopathy is a leading cause of heart failure in diabetes. At the cellular level, diabetic cardiomyopathy leads to altered mitochondrial energy metabolism and cardiomyocyte ultrastructure. We combined electron microscopy and computational modelling to understand the impact of diabetes induced ultrastructural changes on cardiac bioenergetics. We collected transverse micrographs of multiple control and type I diabetic rat cardiomyocytes using electron microscopy. Micrographs were converted to finite element meshes, and bioenergetics was simulated over them using a biophysical model. The simulations also incorporated depressed mitochondrial capacity for oxidative phosphorylation and creatine kinase reactions to simulate diabetes induced mitochondrial dysfunction. Analysis of micrographs revealed a 14% decline in mitochondrial area fraction in diabetic cardiomyocytes, and an irregular arrangement of mitochondria and myofibrils. Simulations predicted that this irregular arrangement, coupled with depressed activity of mitochondrial creatine kinase enzymes, leads to large spatial variation in ADP/ATP profile of diabetic cardiomyocytes. However, when spatially averaged, myofibrillar ADP/ATP ratios of a cardiomyocyte do not change with diabetes. Instead, average concentration of inorganic phosphate rises by 40% due to lower mitochondrial area fraction and dysfunction in oxidative phosphorylation. These simulations indicate that a disorganized cellular ultrastructure negatively impacts metabolite transport in diabetic cardiomyopathy.

cell biology↗

bioRxiv2021

MeasureIce: Accessible on-the-fly measurement of ice thickness in cryo-electron microscopy

Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo electron microscopy. The amorphous atomic structure of the ice that stabilizes and protects biological samples in cryo-EM grids also imprints some addition noise in the TEM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the "ice-channel" technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin sample, achieving a 1.88 [A]ngstrom resolution in subsequent refinement of the atomic map.

biophysics↗

bioRxiv2020

Novel virus-like particle vaccine encoding the circumsporozoite protein of Plasmodium falciparum is immunogenic and induces functional antibody responses

RTS,S is the leading malaria vaccine in development, but has demonstrated only moderate protective efficacy in clinical trials. RTS,S is a virus-like particle (VLP) that uses the human hepatitis B virus as scaffold to display the malaria sporozoite antigen, circumsporozoite protein (CSP). Particle formation requires fourfold excess scaffold antigen, and as a result, CSP represents only a small portion of the final vaccine construct. Alternative VLP or nanoparticle platforms that reduce the amount of scaffold antigen and increase the amount of the target CSP antigen present in particles may enhance vaccine immunogenicity and efficacy. Here, we describe the production and characterization of a novel VLP that uses the small surface antigen (dS) of duck hepatitis B virus to display CSP. The CSP-dS fusion protein successfully formed VLPs without the need for excess scaffold antigen, and thus CSP represented a larger portion of the vaccine construct. Importantly, this is the first report of a dS-based vaccine that formed particles without excess scaffold protein. CSP-dS formed large particles approximately 31-74 nm in size and were confirmed to display CSP on the surface. The CSP-dS VLP was highly immunogenic in mice and induced antibodies to multiple regions of CSP, even when administered at a lower vaccine dosage. Vaccine-induced antibodies demonstrated functional activity, including the ability to interact with complement and Fc{gamma}-receptors, both previously identified as important in malaria immunity. Our novel platform to produce VLPs without excess scaffold protein has wide implications for the future development of innovative vaccines for malaria and other infectious diseases.

immunology↗

bioRxiv2019

Identification of two independent COL5A1 variants in dogs with Ehlers Danlos syndrome

BACKGROUNDThe Ehlers Danlos syndromes (EDS) are a heterogeneous group of heritable disorders affecting connective tissues. The mutations causing the various forms of EDS in humans are well characterized, but the genetic mutations causing EDS-like clinical pathology in dogs are not known, thus hampering accurate clinical diagnosis.\n\nRESULTSClinical analysis of two independent cases of skin hyperextensibility and fragility, one with pronounced joint hypermobility was suggestive of EDS. Whole genome sequencing revealed de novo mutations of COL5A1 in both cases, confirming the diagnosis of the classical form of EDS. The heterozygous COL5A1 p.Gly1013ValfsTer260 mutation characterized in case 1 introduced a premature termination codon and would be expected to result in 1(V) mRNA nonsense-mediated mRNA decay and collagen V haploinsufficiency. While mRNA was not available from this dog, biochemical analysis of the dermis suggested reduced collagen V in the dermis and ultrastructural analysis demonstrated variability in collagen fibril diameter and the presence of collagen aggregates, termed collagen cauliflowers, consistent with COL5A1 mutations underlying classical EDS. In the second case DNA sequencing demonstrated a p.Gly1571Arg missense variant in the COL5A1 gene. While samples were not available for further analysis, such a glycine substitution would be expected to destabilize the strict molecular structure of the collagen V triple helix and thus affect protein stability and/or integration of the mutant collagen into the collagen V/collagen I heterotypic dermal fibrils.\n\nCONCLUSIONSThis is the first report of genetic variants in the COL5A1 gene causing the clinical presentation of EDS in dogs. These data provide further evidence of the important role of collagen V in dermal collagen fibrillogenesis. Importantly from the clinical perspective we show the utility of DNA sequencing, combined with the established clinical criteria, in the accurate diagnosis of EDS in dogs.

genetics↗

bioRxiv2019

Display of malaria transmission-blocking antigens on chimeric DHBV-derived virus-like particles produced in Hansenula polymorpha

1.BackgroundMalaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates.\n\nMethodsWe developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens.\n\nResultsIn this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90 % on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP.\n\nConclusionThe incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.

synthetic biology↗

bioRxiv2018

Insights on the impact of mitochondrial organisation on bioenergetics in high-resolution computational models of cardiac cell architecture

Recent electron microscopy data have revealed that cardiac mitochondria are not arranged in crystalline columns, but are organised with several mitochondria aggregated into columns of varying sizes often spanning the cell cross-section. This raises the question - how does the mitochondrial arrangement affect the metabolite distributions within cardiomyocytes and their impact on force dynamics? Here we employed finite element modelling of cardiac bioenergetics, using computational meshes derived from electron microscope images, to address this question. Our results indicate that heterogeneous mitochondrial distributions can lead to significant spatial variation across the cell in concentrations of inorganic phosphate, creatine (Cr) and creatine phosphate (PCr). However, our model predicts that sufficient activity of the creatine kinase (CK) system, coupled with rapid diffusion of Cr and PCr, maintains near uniform ATP and ADP ratios across the cell cross sections. This homogenous distribution of ATP and ADP should also evenly distribute force production and twitch duration with contraction. These results suggest that the PCr shuttle, and associated enzymatic reactions, act to maintain uniform force dynamics in the cell despite the heterogeneous mitochondrial organization. However, our model also predicts that under hypoxia - activity of mitochondrial CK enzyme and diffusion of high-energy phosphate compounds may be insufficient to sustain uniform ATP/ADP distribution and hence force generation.

systems biology↗

bioRxiv2018

An Automated Workflow for Segmenting Single Adult Cardiac Cells from Large-Volume Serial Block-Face Scanning Electron Microscopy Data

This paper presents a new algorithm to automatically segment the myofibrils, mitochondria and nuclei within single adult cardiac cells that are part of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The algorithm only requires a set of manually drawn contours that roughly demarcate the cell boundary at routine slice intervals (every 50th, for example). The algorithm correctly classified pixels within the single cell with 97% accuracy when compared to manual segmentations. One entire cell and the partial volumes of two cells were segmented. Analysis of segmentations within these cells showed that myofibrils and mitochondria occupied 47.5% and 51.6% on average respectively, while the nuclei occupy 0.7% of the cell for which the entire volume was captured in the SBF-SEM dataset. Mitochondria clustering increased at the periphery of the nucleus region and branching points of the cardiac cell. The segmentations also showed high area fraction of mitochondria (up to 70% of the 2D image slice) in the sub-sarcolemmal region, whilst it was closer to 50% in the intermyofibrillar space. We finally demonstrate that our segmentations can be turned into 3D finite element meshes for cardiac cell computational physiology studies. We offer our large dataset and MATLAB implementation of the algorithm for research use at www.github.com/CellSMB/sbfsem-cardiac-cell-segmenter/. We anticipate that this timely tool will be of use to cardiac computational and experimental physiologists alike who study cardiac ultrastructure and its role in heart function.

bioengineering↗