Biology subjects

Ziegler, C.

Publications and source records attributed to Ziegler, C..

15 recordsLinked to original sources

bioRxiv2026

Connectomics by Sequencing Reveals Self-Organizing Principles of Neuronal Networks in Cerebral Organoids

Functional neural networks emerge as developing neurons form synaptic connections. Revealing how these connections are organized and perturbed in disease requires linking single-neuron connectivity to molecular state across thousands of networks, which is challenging with current methods. We developed COSCO (Connectomics by Sequencing in Cerebral Organoids), combining barcoded monosynaptic rabies tracing with single-cell RNA sequencing to reconstruct over 10,000 networks across organoids. We demonstrate that organoid circuitry self-organizes into stereotyped network motifs governed by cell-intrinsic wiring rules aligning with features of mouse cortical connectivity. Projecting network topology onto transcriptomic space reveals that wiring correlates with cell-adhesion and synaptic gene expression programs. In a Tuberous Sclerosis Complex model, pathological hyperexcitability coincides with topological rewiring: mutant networks become more heterogeneous, driven by the transcriptional distribution of upper layer excitatory cell states. This work establishes cerebral organoids as a model to study self-organization of human connectivity, and its disruption in neurodevelopmental disease. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/755792v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@50ade8org.highwire.dtl.DTLVardef@c97320org.highwire.dtl.DTLVardef@1a53a47org.highwire.dtl.DTLVardef@9520ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Ion channel function of polycystin-2/polycystin-1 heteromer revealed by structure-guided mutagenesis

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations affecting polycystin-1 (PC1) or polycystin-2 (PC2). Recent structural data suggest that PC1 and PC2 can form heterotetrameric ion channels with a 3:1 stoichiometry, with the channel in a closed state. In this hetero-oligomeric formation three PC1 residues (R4100, R4107, and H4111) would work together with two PC2 residues (L677, N681) to block the ion permeation pathway. Here, we provide functional evidence supporting this model. When the pore-blocking residues are replaced by alanines, there is a significant gain-of-function (GOF) effect on PC2/PC1 currents. These PC2/PC1 GOF channels show different selectivity for monovalent cations and reduced Ca2+ permeability compared to PC2 homomers. By varying the expression ratio of PC1 and PC2, we found that PC2 preferentially forms heteromeric complexes with PC1 over homomers. Additionally, a re-interpretation of published PC2/PC1 cryo-electron microscopy data, combined with cysteine modification experiments, suggests that the pore-forming domain of PC1 adopts a canonical TRP-like conformation. The novel PC2/PC1 GOF construct offers a promising approach to investigate the functional impact of mutations linked to ADPKD.

biophysics↗

bioRxiv2024

Relating GPCR domains with functionality: receptor helix-bundle and C-terminus differentially influence GRK-specific functions and β-arrestin-mediated regulation

G protein-coupled receptors (GPCRs) orchestrate diverse physiological responses via intracellular signaling through G proteins, GPCR kinases (GRKs), and arrestins. While the role of G proteins in receptor signaling is well-established, the contributions of GRKs and arrestins remain incompletely understood. Here, we investigate the influence of arrestin-interacting GPCR domains (helix-bundle/C-terminus) on {beta}-arrestin conformations and functions using refined biosensors and advanced cellular knockout systems. By focusing on prototypical class A (b2AR) and B (V2R) receptors and their chimeras (b2V2/V2b2), we can now characterize differential {beta}-arrestin conformational changes as primarily mediated by the receptor C-terminus or helix-bundle. Moreover, we demonstrate that some {beta}-arrestin-supported processes are governed by distinct receptor domains, such as ERK1/2 activation (helix-bundle) and arrestin co-internalization (C-terminus), while others depend on the overall GPCR configuration, such as receptor internalization. Our findings elucidate how individual GPCR domains dictate downstream signaling events, shedding light on the structural basis of receptor-specific signaling and regulation.

pharmacology and toxicology↗

bioRxiv2022

Autoregulation of a trimeric transporter involves the cytoplasmic domains of both adjacent subunits

Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used {micro}s-scale molecular simulations of the BetP trimer to compare reported conformations of the 45 amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from EM data represents a stable global orientation of the C-terminal segment under downregulating conditions. The simulations also allow a detailed molecular description of the C-terminal tail dynamics as well as its interactions with lipids, potassium ions, and the cytoplasmic surface of neighboring transporter subunits. Nevertheless, they do not provide information on the N-terminal segment, whose structure was not resolved by the structural studies. We therefore examined the possible interactions of the N-terminal tail by generating de novo models of its structure in the context of the EM-derived structure. The resultant full-length models of the BetP trimer provide a molecular framework for the arrangement of the terminal domains in the downregulated protein. In this framework, each C-terminal tail contacts the neighboring protomer in the clockwise direction (viewed from the cytoplasm), while the N-terminal tails only contact the protomer in the counterclockwise direction. This framework indicates an intricate interplay between the three protomers of BetP and, specifically, a multi-directionality that may facilitate autoregulation of betaine transport.

biochemistry↗

bioRxiv2022

Osmotic stress response in BetP: How lipids and K+ team up to overcome downregulation

The trimeric betaine symporter BetP senses an osmotic upshift via its osmosensory C-terminal domain and responds rapidly with a steep increase in transport rate. Full activation requires both an elevated internal K+ concentration and a yet unknown membrane stimulus. Moreover, the molecular mechanisms of stress sensing and upregulation remain unclear. Here, we show that K+ binding to BetP in vivo is highly cooperative. Using X-ray crystallography, we identify four putative K+ interaction sites at the C-terminal domains of BetP. Single particle CryoEM on BetP reconstituted in amphipols (AMP-BetP), in the absence of K+, revealed that the three C-terminal domains are oriented symmetrically near the membrane surface, representing a new downregulated state. The presence of K+ resulted in asymmetric partial unfolding of the C-terminal domains, which was assigned as an intermediate between the downregulated state and the conformation observed in crystal structures. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in 2D crystals of BetP reveal glutamate/aspartate and tyrosine responses to K+, in agreement with the identified K+ interaction sites, as well as specific unfolding events in the C-terminal domain upon activation. A rearrangement of the relative protomer orientations confers upregulation through key structural elements involved in the alternating access of BetP affecting sodium and betaine binding affinities. Although K+-specific regulation is unique to BetP we discuss unfolding/refolding of sensory domains as a unifying element in hyperosmotic stress response of osmoregulated transporters.

biophysics↗

bioRxiv2022

VanRS and CroRS cross-talk revealed by coevolutionary modeling regulates antibiotic resistance in VanA-type vancomycin-resistant Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen that can cause bacteremia and endocarditis. Previous studies have shown that concurrent treatment with cephalosporin and vancomycin antibiotics exhibit synergy in vancomycin-resistant E. faecalis to render the bacterium susceptible to antibiotic treatment whereas treatment with each antibiotic separately was not successful. Proteins responsible for mediating vancomycin and cephalosporin resistance are classified as two-component systems (TCS). TCS consist of a histidine kinase that phosphorylates a response regulator after environmental activation. These signaling networks have been shown to exhibit cross-talk interactions, and through direct coupling analysis, we identify encoded specificity between vancomycin resistance TCS, which are horizontally acquired, and cephalosporin resistance TCS, which are endogenous to E. faecalis. To verify cross-talk between these pathways is responsible for vancomycin and cephalosporin synergy, we use RNA-Seq to identify differentially expressed genes in VanA- and VanB-type vancomycin resistant enterococci after treatment with the cephalosporin antibiotic, ceftriaxone, and also with vancomycin. We find that cross-talk between VanSA and CroR in strain HIP11704 may be responsible for synergy, demonstrating that horizontally acquired TCS can have large impacts on pre-existing signaling networks. The presence of encoded specificity between exogenous TCS and endogenous TCS show that the systems co-evolve, and cross-talk between these systems may be exploited to engineer genetic elements that disrupt antibiotic resistance TCS pathways. Author SummaryBacteria may transmit genetic elements to other bacteria through the process known as horizontal gene transfer. In some enterococci, vancomycin resistance genes are acquired this way. Proteins encoded within the bacterial genome can interact with proteins acquired through horizontal gene transfer. The interaction that occurs between proteins VanSA and VanRA is known to mediate vancomycin antibiotic resistance in VanA-type vancomycin resistant enterococci (VRE), and the interaction between proteins CroS and CroR is an important pathway in cephalosporin antibiotic resistance. We show that the VanSA, which is obtained through horizontal gene transfer, inhibits CroR under treatment with antibiotics vancomycin and ceftriaxone. This interaction is responsible for the observed synergy between vancomycin and ceftriaxone in VanA-type VREs. These findings demonstrate how horizontally acquired genes may produce proteins that interrupt known protein interactions, including antibiotic resistance signaling pathways in bacteria. Furthermore, the specific mechanism found for VanA-type VREs provides a basis for engineering of horizontally acquired proteins that disrupt antibiotic resistance pathways.

microbiology↗

bioRxiv2021

The structure of HiSiaQM defines the architecture of tripartite ATP-independent periplasmic (TRAP) transporters

Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria and archaea and provide important uptake routes for many metabolites 1-3. They consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains) that form a functional unit 4. While the structures of the P-domains are well-known, an experimental structure of any QM-domain has been elusive. HiSiaPQM is a TRAP transporter for the monocarboxylate sialic acid, which plays a key role in the virulence of pathogenic bacteria 5. Here, we present the first cryo-electron microscopy structure of the membrane domains of HiSiaPQM reconstituted in lipid nanodiscs. The reconstruction reveals that TRAP transporters consist of 15 transmembrane helices and are structurally related to elevator-type transporters, such as GltPh and VcINDY 6, 7. Whereas the latter proteins function as multimers, the idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. Structural and mutational analyses together with an AlphaFold 8 model of the tripartite (PQM) complex reveal the structural and conformational coupling of the substrate-binding protein to the transporter domains. Furthermore, we characterize high-affinity VHHs that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake in vivo. Thereby, they also confirm the orientation of the protein in the membrane. Our study provides the first structure of any binding-protein dependent secondary transporter and provides starting points for the development of specific inhibitors.

biochemistry↗

bioRxiv2021

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

immunology↗

bioRxiv2021

Quantification and uncertainty of root growth stimulation by elevated CO2 in mature temperate deciduous forest

Increasing CO2 levels are a major global challenge, and the extent to which increasing anthropogenic CO2 emissions can be mitigated by natural carbon sinks remains poorly understood. The uptake of elevated CO2 (eCO2) by the terrestrial biosphere, and subsequent sequestration as biomass in ecosystems, may act as a negative feedback in the carbon budget, but remains hard to quantify in natural ecosystems. Here, we combine large-scale field observations of fine root stocks and flows, derived from belowground imaging and soil cores, with image analysis, stochastic modelling, and statistical inference, to elucidate belowground root dynamics in a mature temperate deciduous forest under free-air CO2 enrichment to 150ppm above ambient levels. Using over 67k frames of belowground observation, we observe that eCO2 leads to relatively faster root production (a peak volume fold change of 4.52 {+/-} 0.44 eCO2 versus 2.58 {+/-} 0.21 control). We identify an increase in existing root elongation relative to root mass decay as the likely causal mechanism for this acceleration. Direct physical analysis of biomass and width measurements from 552 root systems recovered from soil cores support this picture, with lengths and widths of fine roots significantly increasing under eCO2. We use dynamic measurements to estimate fine root contributions to net primary productivity, finding an increase under eCO2, with an estimated mean annual 204 {+/-} 93 g dw m-2yr-1 eCO2 versus 140 {+/-} 60 g dw m-2 yr-1 control. We also quantify and discuss the uncertainties in such productivity measurements. This multi-faceted approach thus sheds quantitative light on the challenging characterisation of the eCO2 response of root biomass in mature temperate forests.

plant biology↗

bioRxiv2021

Aggregated Mycobacterium tuberculosis enhances the inflammatory response

Mycobacterium tuberculosis (Mtb) readily aggregates in culture and Mtb aggregates in the lung were observed in experimental Mtb infection. However, the physiological consequences of Mtb aggregation are incompletely understood. Here we examined the human macrophage transcriptional response to aggregated Mtb relative to infection with non-aggregated single or multiple bacilli per host cell. Infection with aggregated Mtb led to an early upregulation of pro-inflammatory associated genes and enhanced TNF signaling via the NF{kappa}B pathway. Both these pathways were significantly upregulated relative to infection with single bacilli, and TNF signaling was also significantly elevated relative to infection with multiple non-aggregated Mtb. Secretion of TNF and downstream cytokines were also enhanced. On a longer timescale, aggregate infection led to overall increased acidification per macrophage and a high proportion of death in these cells after aggregate phagocytosis. Host cell death did not occur when Mtb aggregates were heat killed despite such clumps being readily picked up. To validate that Mtb aggregates do occur in the human lung, we document Mtb aggregates surrounding a cavity in a human TB lesion. Aggregates may therefore be present in some lesions and elicit a stronger inflammatory response resulting in recruitment of additional phagocytes and their subsequent death, potentially leading to necrosis and transmission.

microbiology↗

bioRxiv2021

GRK2/3/5/6 knockout: The impact of individual GRKs on arrestin-binding and GPCR regulation

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and represent major drug targets. Upon ligand stimulation, GPCRs activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and formation of receptor-arrestin complexes. For many GPCRs, this mechanism triggers receptor desensitisation, internalisation, and possibly a second intracellular signalling wave. Here we created eleven different HEK293 knockout cell clones for GRK2, 3, 5, and 6 individually and in combination. These include four single, two double, four triple, and the quadruple GRK knockout. The statistical evaluation of {beta}-arrestin1/2 interactions for twelve different receptors grouped the tested GPCRs into two main subsets: those for which {beta}-arrestin interaction was mediated by either GRK2, 3, 5, or 6 and those that are mediated by GRK2 or 3 only. Interestingly, the overexpression of specific GRKs was found to induce a robust, ligand-independent {beta}-arrestin interaction with the V2R and AT1R. Finally, using GRK knockout cells, PKC inhibitors, and {beta}-arrestin mutants, we present evidence for differential AT1R-{beta}-arrestin2 complex configurations mediated by selective engagement of PKC, GRK2, or GRK6. We anticipate our novel GRK-knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and {beta}-arrestin complex formation.

cell biology↗

bioRxiv2020

CopR, a global regulator of transcription to maintain copper homeostasis in Pyrococcus furiosus

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analysed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterise the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 M copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the complex regulatory network that enables Pyrococcus to respond to increased copper concentrations.

microbiology↗

bioRxiv2020

Development and validation of IMMUNO-COV: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2

We here describe the development and validation of IMMUNO-COV, a high-throughput clinical test to quantitatively measure SARS-CoV-2-neutralizing antibodies, the specific subset of anti-SARS-CoV-2 antibodies that block viral infection. The test measures the capacity of serum or purified antibodies to neutralize a recombinant Vesicular Stomatitis Virus (VSV) encoding the SARS-CoV-2 spike glycoprotein. This recombinant virus (VSV-SARS-CoV-2-S-{Delta}19CT) induces fusion in Vero cell monolayers, which is detected as luciferase signal using a dual split protein (DSP) reporter system. VSV-SARS-CoV-2-S-{Delta}19CT infection was blocked by monoclonal -SARS-CoV-2-spike antibodies and by plasma or serum from SARS-CoV-2 convalescing individuals. The assay exhibited 100% specificity in validation tests, and across all tests zero false positives were detected. In blinded analyses of 230 serum samples, only two unexpected results were observed based on available clinical data. We observed a perfect correlation between results from our assay and 80 samples that were also assayed using a commercially available ELISA. To quantify the magnitude of the anti-viral response, we generated a calibration curve by adding stepped concentrations of -SARS-CoV-2-spike monoclonal antibody to pooled SARS-CoV-2 seronegative serum. Using the calibration curve and a single optimal 1:100 serum test dilution, we reliably measured neutralizing antibody levels in each test sample. Virus neutralization units (VNUs) calculated from the assay correlated closely (p < 0.0001) with PRNTEC50 values determined by plaque reduction neutralization test against a clinical isolate of SARS-CoV-2. Taken together, these results demonstrate that the IMMUNO-COV assay accurately quantitates SARS-CoV-2 neutralizing antibodies in human sera and therefore is a potentially valuable addition to the currently available serological tests. The assay can provide vital information for comparing immune responses to the various SARS-CoV-2 vaccines that are currently in development, or for evaluating donor eligibility in convalescent plasma therapy studies.

immunology↗

bioRxiv2019

FluidFM as a Tool to Study Adhesion Forces of Bacteria - Optimization of Parameters and Comparison to Conventional Bacterial Probe Scanning Force Spectroscopy

The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells, we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell.

biophysics↗

bioRxiv2018

Assignment of the resting state of the secondary active transporter BetP by integrating spectroscopic measurements and molecular simulations

The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium glutamicum, a soil bacterium used extensively in biotechnology. Although BetP is a homotrimer, biochemical studies have shown that each protomer is able to transport its substrate independently. Crystallographic structures of BetP have been determined in several conformations, seemingly capturing outward-open, inward-open and occluded states, both loaded with the substrate and in the apo form. However, it has been challenging to establish a correspondence between each of these structures and specific states in the mechanism of the transporter under more physiological conditions. To this end, we examined the dynamics of spin-labelled BetP using pulsed electron-electron double resonance (PELDOR) under different stimuli. We then carried out molecular simulations of structures of the BetP monomer to interpret the PELDOR data, using the enhanced-sampling methodology EBMetaD (1), whereby the dynamics of the protein are minimally biased so as to reproduce the experimental data. Comparison of the magnitude of the biasing work required for different input structures permitted us to assign them to specific states of the transport cycle under each of the experimental conditions. In particular, this analysis showed that BetP adopts inward-facing conformations in the presence of excess sodium, and a mixture of states when betaine is added. These studies better delineate the major conformations adopted by BetP in its transport cycle, and therefore provide important insights into its mechanism. More broadly, we illustrate how integrative simulations can aid interpretation of ambiguous structural and spectroscopic data on membrane proteins.\n\nAbbreviations

biophysics↗