Biology subjects

Rasmussen, T.

Publications and source records attributed to Rasmussen, T..

13 recordsLinked to original sources

bioRxiv2026

The Structure of Escherichia coli MscL and its dimer formation in Nanodiscs

Mechanosensitive channels of large conductance (MscL) are essential bacterial safety valves that prevent osmotic lysis by releasing solutes in response to membrane tension. Despite extensive functional studies on Escherichia coli MscL (EcMscL), its high-resolution structure remained unknown. Using cryo-electron microscopy, we present an experimental structure of EcMscL reconstituted in nanodiscs at 3.1 [A] resolution. The structure reveals a pentameric assembly with a narrow hydrophobic gate at the cytosolic side and a periplasmic cavity, consistent with the canonical MscL-fold. Differences to earlier published crystal structures of MscL from other organisms are in the less conserved periplasmic loop. We observe a previously unreported dimeric association of EcMscL pentamers, mediated by residues 61-63 in the periplasmic loop. This dimeric interface is located at the periplasmic side and provides a structural basis for the formation of higher-order clusters. The observed arrangement enables a fluid-like, mosaic packing of channels with center-to-center distances of 5.9-9 nm, consistent with biophysical and imaging data. These findings provide a structural framework for understanding cluster organization of EcMscL that modulates its activity in cellular stress response.

molecular biology↗

bioRxiv2025

SLC26A11 is an atypical solute carrier with dual transport-channel function mediating lysosomal sulfate transport

Membrane transporters and channels are generally assumed to be based on distinct structural and functional principles. SLC26A11, a solute carrier with high expression levels in the brain, has been proposed to function as either an anion transporter or a channel. Here, we resolve this apparent discrepancy by demonstrating that SLC26A11 is a dual-function protein capable of operating as both a sulfate transporter and a chloride channel. By resolving its structure and combining biochemical studies and molecular dynamics simulations, we show that SLC26A11 exhibits all the hallmarks of a secondary transporter. The mechanistic basis for its selective ion transport identifies the protein as the elusive lysosomal sulfate exporter. Additionally, we demonstrate that SLC26A11 exhibits an uncoupled, channel-like chloride conductance gated by proton:sulfate symport. Our finding that the chloride-conducting state arises from the transport cycle may contribute to the development of novel therapeutic strategies for treating brain edema, and the identification of its role in lysosome sulfate efflux may provide new approaches to study and treat lysosomal storage diseases.

biochemistry↗

bioRxiv2024

YnaI exemplifies the diversity of structural gating mechanisms in mechanosensitive channels of small conductance

Osmotically varying environments are challenging for bacterial cells. Sudden drops in osmolytes cause an increased membrane tension and rupture the cells in the absence of protective mechanisms. One family of protective proteins are mechanosensitive channels of small conductance that open in response to membrane tension. Although these channels have a common architecture, they vary widely in the number of transmembrane helices, conductivity, and gating characteristics. Despite of several structures of channels in the open and closed state, the underlying common principles of the gating mechanism are not well understood. Here we show that YnaI opens by radial relocation of the transmembrane sensor paddles together with a shortening of the pore. This contrasts the prototypic smaller MscS which tilts the sensor paddles and retains the pore length. A chimera of both channels with the YnaI sensor paddles and the pore containing C-terminal part of MscS has the conductivity of the pore donor and the tension response of the paddle donor together with the conformational changes of the respective donor. Our research shows that elements with different types of structural rearrangements can be mixed and matched within one channel as long as they support the common area expansion on the periplasmic side.

biochemistry↗

bioRxiv2024

On the neural substrates of mind wandering and dynamic thought: A drug and brain stimulation study

The impact of mind wandering on our daily lives ranges from diminishing productivity, to facilitating creativity and problem solving. There is evidence that distinct internal thought types can be modulated by transcranial direct current stimulation (tDCS), although little is known about optimal stimulation parameters or the mechanisms behind such effects. In addition, recent findings suggest changes in dopamine availability may alter the effect tDCS has on neural and behavioural outcomes. Dopaminergic functioning has also been implicated in executive processes anticorrelated with mind wandering such as attention and working memory, however the neurochemical mechanisms involved in internal thoughts are largely unknown. Here, we investigated the role of dopamine, and tDCS, on internal thought processes. Specifically, using an attentional control task, we tested whether dopamine availability (levodopa or placebo) mediated the effects of online high definition tDCS (HD-tDCS; 2mA, or sham). There was no evidence for our hypothesised effect of left prefrontal cortex HD-tDCS reducing task unrelated thought, nor freely moving thought. This failure to replicate previous HD-tDCS findings emphasises the importance of employing robust methodological practices within this field to improve confidence in the findings. However, we did find that levodopa reduced freely moving thought, relative to placebo. We also found preliminary evidence that dopamine availability may moderate the relationship between stimulation and behavioural variability performance during periods of task unrelated thought. Overall, these findings suggest that stimulation does not affect dynamic internal thought, however there is initial evidence for the potential effectiveness of targeting the dopaminergic system to reduce spontaneous internal thoughts and improve behavioural performance.

neuroscience↗

bioRxiv2024

A filamentous scaffold for gene regulation

Proteins of the Drosophila behaviour/human splicing (DBHS) family are involved in many aspects of gene regulation and maintenance like transcription, splicing and DNA repair. The three known members of this family in humans, Non-POU domain-containing octamer-binding protein (NONO), splicing factor proline/glutamine rich (SFPQ), and paraspeckle protein component 1 (PSPC1), form homo- and heterodimers to fulfil these functions by mediating contacts between RNA, DNA, and other protein factors. The dimers can further dynamically oligomerise through -helical coiled-coils to larger aggregates, which is crucial for many functions of DBHS proteins. While the atomic structures of the dimers are established, the native arrangement in higher oligomers was unknown. Here we present the structure of a filamentous NONO/SFPQ heterooligomer from Cricetulus griseus resolved by cryo-EM. Globular heterodimer domains are alternating on both sides of a strand that is stabilized by an interdigitating network of coiled-coil interactions. Two of these strands assemble into a double strand with only few interactions between them. The globular domains of SFPQ face the counter strand and form a groove while those of NONO face outwards. The different environments of NONO and SFPQ in the filament provide the basis for a differential functionality. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/617013v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@19af4caorg.highwire.dtl.DTLVardef@3d04daorg.highwire.dtl.DTLVardef@9bdc7org.highwire.dtl.DTLVardef@18a6f88_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Lymphostatin: Structure of a large multi-functional virulence factor

Enteropathogenic and Enterohaemorrhagic Escherichia coli are enteric pathogens of global importance and human infections can be life-threatening. Lymphostatin is a key virulence factor of these bacteria, being required for intestinal colonisation and a potent inhibitor of the mitogen- and antigen-activated proliferation of lymphocytes and proinflammatory responses. In some strains, it also mediates adherence to host cells and influences actin nucleation at sites of attachment. This 365 kDa protein requires glycosyltransferase and cysteine protease motifs for activity against lymphocytes, but high-resolution structural information has proven elusive and the molecular mechanisms by which it acts remain unclear. Here, we describe the structure of lymphostatin from the prototype O127:H6 enteropathogenic E. coli strain determined by electron cryo-microscopy. Our results reveal two glycosyltransferase domains, one PaTox-like protease domain, an ADP-ribosyltransferase domain, and a delivery domain. Long linkers act to hold these domains together. These linkers occlude the catalytic sites of the N-terminal glycosyltransferase and protease domains. In this dormant state, lymphostatin binds to HEK-293T cells, where it forms large clusters before being taken up and sequestered into cytosolic foci. With more functional domains than any other known large bacterial toxin, lymphostatin can be regarded as the multifunctional Swiss army knife of pathogenic Escherichia coli, enabling complex interactions with the host cells in different environments.

biophysics↗

bioRxiv2023

On the role of prefrontal and parietal cortices in mind wandering and dynamic thought

Mind wandering is a common phenomenon in our daily lives and can have both an adaptive and detrimental impact. Recently, a dynamic framework has been proposed to characterise the heterogeneity of internal thoughts, suggesting there are three distinct thought types which can change over time - freely moving, deliberately constrained, and automatically constrained (thoughts). There is very little evidence on how different types of dynamic thought map onto the brain. Previous research has applied non-invasive transcranial direct current stimulation (tDCS) to causally implicate the prefrontal cortex and inferior parietal lobule in general mind wandering. However, a more recently developed and nuanced technique, high-definition tDCS (HD-tDCS), delivers more focal stimulation able to target specific brain regions. Therefore, the current study investigated the effect of anodal HD-tDCS applied to the left prefrontal and right inferior parietal cortices (with the occipital cortex included as an active control) on mind wandering, and specifically, the causal neural substrates of the three internal dynamic thought types. This was a single session study using a novel task which allows investigation into how dynamic thoughts are associated with behavioural variability and the recruitment of executive control operations across the three brain regions. Anodal stimulation to the prefrontal cortex decreased freely moving thought and anodal stimulation to the parietal lobule decreased deliberately constrained thought, with preliminary evidence for an increase in freely moving thought in the occipital cortex as well. These findings support the heterogenous nature of mind wandering, revealing that different brain regions are implicated in distinct dynamic thought types.

neuroscience↗

bioRxiv2023

Discovery of a functionally selective serotonin 5-HT1A receptor agonist for the treatment of pain

The G protein-coupled serotonin receptor 5-HT1AR mediates antinociception and may serve as a valuable target for the treatment of pain. Starting from a chemical library, ST171, a bitopic chemotype activating 5-HT1AR was evolved. In vitro pharmacological investigations of ST171 revealed potent and selective Gi activation (EC50 = 0.3 nM), with marginal Gs and {beta}-arrestin recruitment. Preclinical studies in mice showed that ST171 was effective in acute and chronic (inflammatory and neuropathic) pain models, without causing sedation. Comparison of cryo-EM structures of a 5-HT1AR-Gi complex bound to the functionally biased agonist ST171, with a structure bound to the functionally balanced agonist befiradol, showed that both ligands bind to the same orthosteric site, but address different exo-sites. The individual poses are associated with ligand-specific helical dispositions and rearrangements of microdomains. Complementation of these studies with molecular dynamics simulations allowed us to derive structural features associated with ST171s functional selectivity, a phenomenon that may be crucial to the discovery of more effective and safe GPCR drugs.

pharmacology and toxicology↗

bioRxiv2022

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. The LipIDens code is open-source and embedded within a notebook format to assist automation and usability.

biophysics↗

bioRxiv2021

Efficient and Rapid generation of neural stem cells by direct conversion Fibroblasts with A Single microRNA

Neural stem cells (NSCs) have great potential in the application of neurodegenerative disease therapy, drug screening and disease modeling. NSC can be generated by reprogramming from terminally differentiated cells with transcription factors or small molecules. However, current methods for producing NSCs involve the danger of integrating foreign genes into the genome and the problem of low efficiency. Here, we report an efficient method to generate NSCs from human skin-derived fibroblasts with microRNA (mir-302a) in 2-3 days. The induced NSCs (iNSCs) have more than 90% of purity. Their morphology is similar to regular NSCs, expressing key markers including Nestin, Pax6 and Sox2, and can be expanded for more than 20 passages in vitro. They can also differentiate into functional neuron progeny, astrocytes and oligodendrocytes as well. Those cells can elicit action potential, can be xeno-transplanted into the brain of immune-deficient mice, and can survive and differentiate in vivo without tumor formation. This study shows that a single part of pluripotency-inducing mir-302 cluster can drive fibroblasts reprogramming, providing a general platform for high-efficiency generation of individual-specific human NSCs for studies of neuron system development and regenerative cell therapy.

neuroscience↗

bioRxiv2019

Effects of mesotocin on social bonding in pinyon jays

The neuropeptide oxytocin influences mammalian social bonding by facilitating the building and maintenance of parental, sexual, and same-sex social relationships. However, we do not know whether the function of the avian homologue mesotocin is evolutionarily conserved across birds. While it does influence avian prosocial behavior, mesotocins role in avian social bonding remains unclear. Here, we investigated whether mesotocin regulates the formation and maintenance of same-sex social bonding in pinyon jays (Gymnorhinus cyanocephalus), a member of the crow family. We formed squads of four individually housed birds. In the first, pair-formation phase of the experiment, we repeatedly placed pairs of birds from within the squad together in a cage for short periods of time. Prior to entering the cage, we intranasally administered one of three hormone solutions to both members of the pair: mesotocin, oxytocin antagonist, or saline. Pairs received repeated sessions with administration of the same hormone. In the second, pair-maintenance phase of the experiment, all four members of the squad were placed together in a large cage, and no hormones were administered. For both phases, we measured the physical proximity between pairs as our proxy for social bonding. We found that, compared to saline, administering mesotocin or oxytocin antagonist did not result in different proximities in either the pair-formation or pair-maintenance phase of the experiment. Therefore, at the dosages and time frames used here, exogenously introduced mesotocin did not influence same-sex social bond formation or maintenance. Like oxytocin in mammals, mesotocin regulates avian prosocial behavior; however, unlike oxytocin, we do not have evidence that mesotocin regulates social bonds in birds.

animal behavior and cognition↗

bioRxiv2018

Merging in-solution X-ray and neutron scattering data allows fine structural analysis of membrane-protein detergent complexes

AbstractIn-solution small angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labelled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. Firstly, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS which has been adapted to predict SANS data. Secondly, the use of MONSA which allows to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC=\"FIGDIR/small/324103_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (67K):\norg.highwire.dtl.DTLVardef@f7e17corg.highwire.dtl.DTLVardef@1f4987dorg.highwire.dtl.DTLVardef@1475c79org.highwire.dtl.DTLVardef@839b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2017

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis

Fungi undergo changes in cell shape in response to environmental stimuli that drive pathogenesis and niche adaptation, such as the yeast-to-hyphal transition of dimorphic fungi in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify bacterial peptidoglycan as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and E. coli co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established and previously undescribed regulators of Titanisation in vitro and investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-titan transition and establish an essential in vitro model for the future characterization of this important morphotype.\n\nAuthor SummaryChanges in cell shape underlie fungal pathogenesis by allowing immune evasion and dissemination. Aspergillus and Candida albicans hyphae drive tissue penetration. Histoplasma capsulatum and C. albicans yeast growth allows evasion and dissemination. As major virulence determinates, morphogenic transitions are extensively studied in animal models and in vitro. The pathogenic fungus Cryptococcus neoformans is a budding yeast that, in the host lung, switches to an unusual morphotype termed the Titan cell. Titans are large, polyploid, have altered cell wall and capsule, and produce haploid daughters. Their size prevents engulfment by phagocytes, yet they are linked to dissemination and altered immune response. Despite their important influence on disease, replicating the yeast-to-Titan switch in vitro has proved challenging. Here we show that Titans are induced by host-relevant stimuli, including serum and bronchio-alveolar lavage fluid. We identify bacterial peptidoglycan as a relevant inducing compound and predict an in vivo Titan defect for a clinical isolate. Genes regulating in vivo Titanisation also influence in vitro formation. Titanisation is a conserved morphogenic switch across the C. neoformans species complex. Together, we show that Titan cells are a regulated morphotype analogous to the yeast-to-hyphal transition and establish new ways to study Titans outside the host lung.

microbiology↗