Biology subjects

Hansen, S. B.

Publications and source records attributed to Hansen, S. B..

17 recordsLinked to original sources

bioRxiv2026

Interdigitating coiled-coil tetramers define the helical architecture of GFAP filaments in astrocytes

GFAP filaments are core elements of the cytoskeleton in astrocytes, shaping cellular processes and influencing central nervous system function and pathology. How these filaments are built at the molecular level has remained unclear. Using cryo-electron microscopy and cryo-electron tomography, we show that GFAP filaments are helical polymers assembled from extended and twisted coiled-coil tetramers that interdigitate to form a complex filament tube. In the filament lumen, the low-complexity head domains aggregate to form a flexible fibre, while the tail domains extend from the surface and facilitate higher-order bundling of the filaments, indicating that both the low-complexity structural regions and the well-ordered coiled-coils of the filament tube contribute to the unique structure of GFAP filaments. Our findings provide molecular insights into the pathogenesis of Alexander Disease, localising multiple deleterious mutations to the critical interlock interaction between successive tetramers emergent in the final filament assembly.

neuroscience↗

bioRxiv2026

Long-term single-molecule Ca2+ flux recordings reveal mode-switching regulation of Ca2+-ATPases

Calcium (Ca{superscript 2}) is a universal second messenger that governs processes ranging from muscle contraction and secretion to gene expression and cell fate. Ca{superscript 2}-ATPases establish and maintain steep Ca{superscript 2} gradients across intracellular membranes, yet how regulatory inputs modulate the underlying single-pump Ca{superscript 2} currents has remained inaccessible. Here we develop a non-saturating, self-regenerating single-vesicle assay that monitors over hours the zeptoampere (10-{superscript 2}{superscript 1} A) currents produced by individual Ca{superscript 2}-ATPases. In parallel, we establish a workflow to record single-molecule currents from human sarco/endoplasmic reticulum Ca{superscript 2}-ATPases (hSERCA) in native endoplasmic reticulum vesicles. Using reconstituted LMCA1, a bacterial SERCA homologue, we observe stochastic switching between minute-long pumping and inactive modes, as well as uncoupled Ca{superscript 2} leakage events that are suppressed by vanadate. Extravesicular pH controls a previously unrecognized dormant pre-activation mode that delays the onset of pumping, without measurably altering pumping rates or active-mode lifetimes. Extending the assay to endogenous hSERCA reveals delayed activation and ultraslow pumping/inactive mode-switching without detectable transprotein Ca{superscript 2} leakage. ATP and Ca{superscript 2} regulate the probability of hSERCA activation by modulating dormant-mode occupancy. Together, these results extend ultraslow mode-switching, previously observed only for proton pumps, to Ca{superscript 2}-ATPases and identify probability-gated entry into productive cycling as a distinct regulatory axis of human Ca{superscript 2}-ATPase regulation that can modulate the timing and heterogeneity of Ca{superscript 2} store refilling without changing on-cycle kinetics.

biophysics↗

bioRxiv2025

Structural basis for size-selective perception of chitin in plants

Plants detect microbes through pattern recognition receptors that perceive conserved microbial surface motifs known as microbe-associated molecular patterns (MAMPs). LysM receptors recognize and mediate downstream responses to chitinous MAMPs. Here, we elucidate the mechanism for the specific recognition of long-chain chitin oligomers and identify a hallmark bridge domain characteristic for the CHIP receptor class. Structural analysis of receptor-ligand complexes, biochemistry, and in planta functional studies using inhibitory nanobodies reveal the mechanism of size-selective, high affinity chitin perception in Lotus japonicus and Medicago truncatula. Additionally, we identify CERKs as low-affinity, yet essential co-receptors and propose a mechanistic model for a ligand-induced core signaling heterocomplex. Our findings provide mechanistic insights into plant chitin perception and the formation of receptor complexes critical for immune signaling.

plant biology↗

bioRxiv2025

Sample-specific CpG loci are important for accurate long-read methylation analysis

Methylated CpG loci mutate at high rates, but it remains challenging to identify sample-specific non-reference CpG loci for methylation analyses. Here, we present a likelihood-based method that identifies sample-specific CpG loci at high precision in long-read sequencing data from Nanopore and PacBio platforms. Inclusion of non-reference CpG loci increased the number of loci and reduced reference bias, and is important for accurate estimation of global methylation levels across samples.

genomics↗

bioRxiv2025

FilaBuster: A Strategy for Rapid, Specific, and Spatiotemporally Controlled Intermediate Filament Disassembly

Intermediate filaments (IFs) play key roles in cellular mechanics, signaling, and organization, but tools for their rapid, selective disassembly remain limited. Here, we introduce FilaBuster, a photochemical approach for efficient and spatiotemporally controlled IF disassembly in living cells. FilaBuster uses a three-step strategy: (1) targeting HaloTag to IFs, (2) labeling with a covalent photosensitizer ligand, and (3) light-induced generation of localized reactive oxygen species to trigger filament disassembly. This modular strategy applies broadly across IF subtypes--including vimentin, GFAP, desmin, peripherin, and keratin 18--and is compatible with diverse dyes and imaging platforms. Using vimentin IFs as a model system, we establish a baseline implementation in which vimentin-HaloTag labeled with a photosensitizer HaloTag ligand triggers rapid and specific IF disassembly upon light activation. We then refine this approach by (i) expanding targeting strategies to include a vimentin nanobody-HaloTag fusion, (ii) broadening the range of effective photosensitizers, and (iii) optimizing irradiation parameters to enable precise spatial control over filament disassembly. Together, these findings position FilaBuster as a robust platform for acute, selective, and spatiotemporally precise disassembly of IF networks, enabling new investigations into their structural and functional roles in cell physiology and disease.

cell biology↗

bioRxiv2025

Multi-domain O-GlcNAcase structures reveal allosteric regulatory mechanisms

Nucleocytoplasmic protein O-GlcNAcylation is an essential modification catalysed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAc hydrolase (OGA), a multi-domain enzyme that also contains a C-terminal pseudo-histone acetyltransferase (pHAT) domain. OGA and OGT are tightly regulated using O-GlcNAc-dependent feedback mechanisms that are largely unknown. Although the structure of the OGA homodimeric catalytic domain has been reported, the structure and function of the pHAT domain remains poorly understood. We describe a crystal structure of the Trichoplax adhaerens pHAT domain and cryo-EM data of the multi-domain T. adhaerens and human OGAs, together with surface plasmon resonance and small-angle X-ray scattering studies. Our findings show that the eukaryotic OGA pHAT domains affect O-GlcNAc homeostasis, and form catalytically incompetent, symmetric homodimers, projecting a partially conserved putative peptide binding site available for interactions with binding partners. While there is evidence for symmetric OGA multi-domain dimers in solution, interactions between the linkers to the pHAT domains allow these to adopt a limited range of positions. In hOGA, the positions of the pHAT domains determine the wider active site environment through a key conformational change involving a tryptophan in a flexible arm region. Taken together, these multi-domain OGA structures reveal allosteric mechanisms of regulation.

molecular biology↗

bioRxiv2024

Two residues reprogram immunity receptor kinases to signal in nitrogen-fixing symbiosis

Receptor signalling determines cellular responses and is crucial for defining specific biological outcomes. In legume root cells, highly similar and structurally conserved chitin and Nod factor receptor kinases activate immune or symbiotic pathways, respectively, upon perception of chitinous ligands. Here, we show that specific amino acid residues in the intracellular part of the Nod factor receptor NFR1 determine signalling specificity and enable the distinction between immune and symbiotic responses. Functional investigation of CERK6, NFR1 and receptor variants hereof revealed a conserved motif that we term Symbiosis Determinant 1 in the juxtamembrane region of the kinase domain that is key for symbiotic signalling. We demonstrate that two residues in Symbiosis Determinant 1 are indispensable hallmarks for NFR1-type receptors and are sufficient to convert Lotus CERK6 and barley RLK4 kinase outputs to enable symbiotic signalling in Lotus japonicus.

plant biology↗

bioRxiv2024

GABA and astrocytic cholesterol determine the lipid environment of GABAAR in cultured cortical neurons.

The {gamma}-aminobutyric acid (GABA) type A receptor (GABAAR), a GABA activated pentameric chloride channel, mediates fast inhibitory neurotransmission in the brain. The lipid environment is critical for GABAAR function. How lipids regulate the channel in the cell membrane is not fully understood. Here we employed super resolution imaging of lipids to demonstrate that the agonist GABA induces a rapid and reversible membrane translocation of GABAAR to phosphatidylinositol 4,5-bisphosphate (PIP2) clusters in mouse primary cortical neurons. This translocation relies on nanoscopic separation of PIP2 clusters and lipid rafts (cholesterol-dependent ganglioside clusters). In a resting state, the GABAAR associates with lipid rafts and this colocalization is enhanced by uptake of astrocytic secretions. These astrocytic secretions enhance endocytosis and delay desensitization. Our findings suggest intercellular signaling from astrocytes regulates GABAAR location based on lipid uptake in neurons. The findings have implications for treating mood disorders associated with altered neural excitability.

neuroscience↗

bioRxiv2024

Structure of a Ca2+ bound phosphoenzyme intermediate in the inward-to-outward transition of Ca2+-ATPase 1 from Listeria monocytogenes

Active transport by Ca2+-ATPases of the P-type ATPase family maintain a very low cytosolic calcium concentration and steep electrochemical gradients. Detailed mechanisms of this transport have been described from structures of mammalian sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) stabilized by inhibitors at specific intermediate steps of the transport cycle. An essentially irreversible step is crucial to prevent reflux in active transport against steep gradients. Single-molecule FRET (smFRET) study of the bacterial Ca2+-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states, suggesting that calcium release from this intermediate is the irreversible step. Here, we present a 3.5[A] cryo-EM structure for a four-glycine insertion mutant (G4-LMCA1) in a lipid nanodisc obtained under turnover conditions and adopting such a calcium-bound intermediate, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain is similar to calcium-bound E1P-ADP like conformation of SERCA. Missing density for the E292 residue at the calcium site (equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism for the inward-to-outward transition in Ca2+-ATPases, where ADP release and re-organisation of the cytoplasmic domains precede calcium release.

molecular biology↗

bioRxiv2023

SAMPLING THE ZEBRAFISH GUT MICROBIOTA - A GENOME RESOLVED METAGENOMIC APPROACH

The zebrafish is a promising model organism in the field of functional microbiota research. However, studies on the functional landscape of the zebrafish gut microbiota through shotgun based metagenomics are scarce. Thus, there is a lack of consensus regarding an appropriate sampling method that accurately represents the zebrafish gut microbiota. To address this, we sought to systematically test and evaluate four different methods of sampling the zebrafish gut microbiota: collection of feces from the tank, the whole gut, intestinal content and the application of ventral pressure to facilitate extrusion of gut material. In addition, we included water samples as an environmental control to address the potential influence of the environmental microbiota on the data interpretation. To compare these sampling methods in a context of microbiota-based studies we employed a combination of genome resolved metagenomics and 16S metabarcoding techniques. We observed differences among sample types on all levels including sampling, bioinformatic processing, metagenome co-assemblies, generation of metagenome-assembled genomes (MAGs), functional potential, MAG coverage and micro-diversity. Furthermore, our comparison to the environmental control demonstrated the potential impact of the environmental contamination on data interpretation. The findings emphasise the importance of considering the choice of sampling method. While all sample types tested are informative about the zebrafish gut microbiota, the optimal sample type depends on the specific objectives of the study.

microbiology↗

bioRxiv2023

Super-resolution imaging of potassium channels with genetically encoded EGFP.

The plasma membrane is a well-organized structure of lipids and proteins, segmented into lipid compartments under 200 nm in size. This specific spatial patterning is crucial for the function of proteins and necessitates super-resolution imaging for its elucidation. Here, we establish that the genetically encoded enhanced green fluorescent protein (EGFP), when combined with direct optical reconstruction microscopy (dSTORM), tracks shear- and cholesterol-induced nanoscopic patterning of potassium channels overexpressed in HEK293T cells. Leveraging EGFP in dSTORM (EGFP-STORM), our findings indicate that cholesterol directs the C-terminus of TWIK-related potassium channel (TREK-1) to ceramide-enriched lipid ganglioside (GM1) clusters. In the absence of the C-terminus, the channel associates with phosphatidylinositol 4,5-bisphosphate (PIP2) cluster. Similarly, cholesterol derived from astrocytes repositions EGFP-tagged inward-rectifying potassium (Kir) channels into GM1 clusters. Without cholesterol, the channel aligns with PIP2 lipids. We deduce that cholesterols interaction with Kir sequesters the channel, separating it from its activating lipid PIP2. Fundamentally, a genetically encoded EGFP tag should make any protein amenable to dSTORM analysis.

cell biology↗

bioRxiv2023

Host-gut microbiota interactions shape parasite infections in farmed Atlantic salmon

Animals and their associated microbiota share long evolutionary histories. Both host genotype and associated microbiota influence phenotypes such as growth and disease resilience. We applied a hologenomic approach to explore the relationship between host and microbiota in shaping lifetime growth and parasitic cestode infection in farmed Atlantic salmon. Genomes, transcriptomes, metabolomes and metagenomes were generated from the guts of 460 harvest-aged salmon, 82% of which were naturally infected with an intestinal cestode. One salmonid-specific Mycoplasma dominated the gut microbiota of uninfected salmon. However, the microbiota was perturbed in smaller, parasitised fish, with increased abundance of Vibrionaceae and other Mycoplasma species previously linked to the cestode microbiota. The cestode-associated Mycoplasma carry more virulence-associated genes than the salmonid Mycoplasma. Colonisation by one cestode-associated Mycoplasma was associated with a region of the salmon genome encoding several long noncoding RNA genes previously associated with host control of intestinal microbiota. Integrating the multiple omic datasets revealed coordinated changes in the salmon gut transcriptome and metabolome that correlated with shifts in the microbiota of smaller, parasitised fish. Our results suggest that cestode infections introduce new microbes and trigger host responses, altering the gut microbiota with increases in potentially pathogenic microbes. Establishment of these microbes is partially shaped by the genetic background of the host. Our study highlights the value of a hologenomic approach for gaining an in-depth understanding of trilateral interactions among host, microbiota and parasite.

systems biology↗

bioRxiv2022

Regulation of neuroinflammation by astrocyte-derived cholesterol.

Neurodegeneration and its concomitant loss of cognitive function are closely linked to neuroinflammation and lipid accumulation, particularly cholesterol. In the brain, astrocytes synthesize cholesterol and deliver it to surrounding cells via apolipoprotein E (apoE). Astrocyte activation increases apoE secretion and promotes neuronal cell death, yet the link between astrocyte-derived cholesterol and inflammation remains unclear. Here we show that pro-inflammatory cytokines stimulate cholesterol synthesis and release from astrocytes. Immune cells take up this cholesterol, which clusters pro-inflammatory receptors in lipid rafts, amplifying inflammatory signaling and gene expression. Astrocyte-specific knockout of cholesterol synthesis (via SREBP2 deletion) reduces inflammatory cytokine production in an Alzheimers disease (AD) mouse model and shortens neuroinflammation triggered by peripheral lipopolysaccharide (LPS) injection. These findings identify astrocyte-derived cholesterol as a paracrine signal that helps drive neuroinflammation in microglia and brain-resident macrophages.

neuroscience↗

bioRxiv2022

An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies have uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 entry and fusion independent of transmembrane protease serine 2 expression in multiple cell types. We also demonstrate a role for ACAT in regulating SARS-CoV-2 RNA replication in primary bronchial epithelial cells. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled in the acute phase of infection. Thus, re-purposing of available ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.

immunology↗

bioRxiv2019

Measuring anesthetic resistance in Drosophila by VAAPR

Volatile anesthetics are compounds which are commonly used to induce a reversable loss of consciousness (LOC) in animals. The molecular mechanism of how anesthetics induce LOC is largely unknown. However, observations have been made which show that there are genetically-encoded traits which influence the effective concentration of anesthetics in the inducement of LOC. Despite this long-term observation, little progress has been made in identifying genes involved in anesthetic sensitivity. One reason for this is that many techniques to test anesthetic sensitivity are technically challenging and are inhibitory for high-throughput studies. Here we introduce a technique for testing volatiles and aerosols with positional recording (VAAPR), a method which allows for high-throughput testing of the effect of anesthetics and other aerosolized drugs using Drosophila. Using VAAPR we show that the enzyme phospholipase D (PLD) significantly shifts the concentration of diethyl ether, chloroform, and isoflurane needed to induce LOC in Drosophila. We also show that PLD is required for a paradoxical hyperactivity phenotype. We expect that this technique will allow for additional genes to be found which control anesthetic sensitivity as well as other behavioral phenotypes.

pharmacology and toxicology↗

bioRxiv2019

Phospholipase D Transduces Force to TREK-1 Channels in a Biological Membrane

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5- bisphosphate (PIP2) clusters. First, mechanical force deforms the ordered lipids, which lowers membrane cholesterol, disrupts the interaction of PLD2 with the GM1 lipids, and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response. SummaryShear thinning activates TREK-1 through a second messenger.

neuroscience↗

bioRxiv2018

Studies on the mechanism of general anesthesia

Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK related K+ channels (TREK-1) and reversibly induce loss of consciousness. For a hundred years anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of palmitate-mediated localization of phospholipase D2 (PLD2) to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in cell patch-clamp. Localization of PLD2 renders the anesthetic-insensitive TRAAK channel sensitive. General anesthetics chloroform, isoflurane, diethyl ether, xenon, and propofol disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLDnull flies resist anesthesia. Our results establish a membrane mediated target of inhaled anesthesia and suggest PA helps set anesthetic sensitivity in vivo.

neuroscience↗