Biology subjects

Weidtkamp-Peters, S.

Publications and source records attributed to Weidtkamp-Peters, S..

11 recordsLinked to original sources

bioRxiv2025

A novel biosensor for ferrous iron developed via CoBiSe:A computational method for rapid biosensor design

Genetically encoded biosensors enable monitoring of metabolite dynamics in living organisms. We present CoBiSe, a computational approach using Constraint Network Analysis to identify optimal insertion sites for reporter modules in molecular recognition elements (MREs). Applied to the iron-binding protein DtxR from Corynebacterium glutamicum, CoBiSe identified a flexible connective loop (residues 138-150) for inserting the reporter module, resulting in IronSenseR, a novel ratiometric biosensor for ferrous iron (Fe{superscript 2}). IronSenseR demonstrates high specificity for Fe{superscript 2} with dissociation constants of 1.55 {+/-} 0.08 {micro}M (FeSO4) and 2.44 {+/-} 0.28 {micro}M (FeCl2), while showing no binding to Fe3 and other divalent cations. In vivo assessment in Escherichia coli, Pseudomonas putida and Corynebacterium glutamicum confirmed IronSenseRs capability to detect changes in the intracellular iron pool. The creation of IronSenseR underlines that, by reducing search space and eliminating labor-intensive screening, CoBiSe streamlines biosensor development and enables precise creation of next-generation biosensors for diverse metabolites.

bioengineering↗

bioRxiv2025

Acylation of the RTX Toxin MbxA stimulates host membrane disruption through a specific interaction with cholesterol

RTX toxins, characterized by their calcium-binding glycine-rich repeat regions, are known for their ability to disrupt host cell membranes. Although the acylation of specific lysine residues in these toxins is established as crucial for their hemolytic activity, the precise mechanisms underlying this enhancement remain unclear. In this study, we explore the role of acylation in the pore-forming behaviour of the RTX toxin MbxA by comparing the lytic activities of acylated MbxA and its non-acylated form, proMbxA, on in vitro membrane systems, as well as living cells. Our findings demonstrate that a cholesterol specific interaction promotes MbxA- induced membrane disruption in an acylation-dependent manner. Further analysis revealed that acylation is not necessary for initial membrane binding but markedly enhances pore formation. Overall, our results offer valuable insights into the molecular determinants that regulate RTX toxin activity, highlighting a complex interplay between lipid composition (sterols), acylation, and membrane disruption, thereby advancing our understanding of RTX toxin pathogenesis.

biochemistry↗

bioRxiv2023

Probing macromolecular crowding at the lipid membrane interface with genetically-encoded sensors

Biochemical processes within the living cell occur in a highly crowded environment. The phenomenon of macromolecular crowding is not an exclusive feature of the cytoplasm and can be observed in the densely protein-packed, nonhomogeneous cellular membranes and at the membrane interfaces. Crowding affects diffusional and conformational dynamics of proteins within the lipid bilayer, and modulates the membrane organization. However, the non-invasive quantification of the membrane crowding is not trivial. Here, we developed the genetically- encoded fluorescence-based sensor for probing the macromolecular crowding at the membrane interfaces. Two sensor variants, both composed of fluorescent proteins and a membrane anchor, but differing by the flexible linker domains were characterized in vitro, and the procedures for the membrane reconstitution were established. Lateral pressure induced by membrane-tethered synthetic and protein crowders altered the sensors conformation, causing increase in the intramolecular Forsters resonance energy transfer. The effect of protein crowders only weakly correlated with their molecular weight, suggesting that other factors, such as shape and charge play role in the quinary interactions. Upon their expression, the designed sensors were localized to the inner membrane of E. coli, and measurements performed in extracted membrane vesicles revealed low level of interfacial crowding. The sensors offer broad opportunities to study interfacial crowding in a complex environment of native membranes, and thus add to the toolbox of methods for studying membrane dynamics and proteostasis.

biophysics↗

bioRxiv2023

Live-cell super-resolution nanoscopy reveals modulation of cristae dynamics in bioenergetically compromised mitochondria

Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here we investigated whether and how cristae membrane remodelling is dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential ({Delta}{Psi}m), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not change the number of cristae remodelling events. CCCP treatment leading to {Delta}{Psi}m abrogation even enhanced the cristae dynamics showing their {Delta}{Psi}m-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange and cristae membrane dynamics. Summary BlurbCristae morphology and dynamics are intricately connected

cell biology↗

bioRxiv2022

Heterologously secreted MbxA from Moraxella bovis induces a membrane blebbing response of the human host cell

Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live- cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

biochemistry↗

bioRxiv2021

mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the hosts intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, to unravel mGBP2 functions, we report Galectin-9 (Gal9) as a critical mGBP2 interaction partner engaged for immunity to T. gondii. Interestingly, Gal9 also accumulates and colocalizes with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that Gal9 is a crucial factor for the mGBP2 coordinated cell autonomous host defense mechanism against T. gondii.

immunology↗

bioRxiv2021

Micro-Meta App: an interactive software tool to facilitate the collection of microscopy metadata based on community-driven specifications

For the information content of microscopy images to be appropriately interpreted, reproduced, and meet FAIR (Findable Accessible Interoperable and Reusable) principles, they should be accompanied by detailed descriptions of microscope hardware, image acquisition settings, image pixel and dimensional structure, and instrument performance. Nonetheless, the thorough documentation of imaging experiments is significantly impaired by the lack of community-sanctioned easy-to-use software tools to facilitate the extraction and collection of relevant microscopy metadata. Here we present Micro-Meta App, an intuitive open-source software designed to tackle these issues that was developed in the context of nascent global bioimaging community organizations, including BioImaging North America (BINA) and QUAlity Assessment and REProducibility in Light Microscopy (QUAREP-LiMi), whose goal is to improve reproducibility, data quality and sharing value for imaging experiments. The App provides a user-friendly interface for building comprehensive descriptions of the conditions utilized to produce individual microscopy datasets as specified by the recently proposed 4DN-BINA-OME tiered-system of Microscopy Metadata model. To achieve this goal the App provides a visual guide for a microscope-user to: 1) interactively build diagrammatic representations of hardware configurations of given microscopes that can be easily reused and shared with colleagues needing to document similar instruments. 2) Automatically extracts relevant metadata from image files and facilitates the collection of missing image acquisition settings and calibration metrics associated with a given experiment. 3) Output all collected Microscopy Metadata to interoperable files that can be used for documenting imaging experiments and shared with the community. In addition to significantly lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training users that have limited knowledge of the intricacies of light microscopy experiments. To ensure wide-adoption by microscope-users with different needs Micro-Meta App closely interoperates with MethodsJ2 and OMERO.mde, two complementary tools described in parallel manuscripts.

bioinformatics↗

bioRxiv2020

Polycistronic gene expression in the model microorganism Ustilago maydis

AbtractEukaryotic microorganisms transcribe monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. Here, we establish such a system in the well-studied model microorganism Ustilago maydis. Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated in vivo using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides in vivo and demonstrated the applicability of 2A peptide technology for U. maydis in basic and applied science.

microbiology↗

bioRxiv2019

PLETHORA and WOX5 interaction and subnuclear localisation regulates Arabidopsis root stem cell maintenance

Maintenance and homeostasis of the stem cell niche (SCN) in the Arabidopsis root is essential for growth and development of all root cell types. The SCN is organized around a quiescent center (QC) maintaining the stemness of cells in direct contact. The key transcription factors (TFs) WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and PLETHORAs (PLTs) are expressed in the SCN where they maintain the QC and regulate distal columella stem cell (CSC) fate. Here, we describe the concerted mutual regulation of the key TFs WOX5 and PLTs on a transcriptional and protein interaction level. Additionally, by applying a novel SCN staining method, we demonstrate that both WOX5 and PLTs regulate root SCN homeostasis as they control QC quiescence and CSC fate interdependently. Moreover, we uncover that some PLTs, especially PLT3, contain intrinsically disordered prion-like domains (PrDs) that are necessary for complex formation with WOX5 and its recruitment to subnuclear microdomains/nuclear bodies (NBs) in the CSCs. We propose that this partitioning of PLT-WOX5 complexes to NBs, possibly by phase separation, is important for CSC fate determination.

plant biology↗

bioRxiv2019

Over the rainbow: a practical guide for fluorophore selection in plant FRET experiments

Receptor-like kinases (RLK) and receptor-like proteins (RLP) often interact in a combinatorial manner depending on tissue identity, membrane domains, or endo- and exogenous cues, and the same RLKs or RLPs can generate different signaling outputs depending on the composition of the receptor complexes they are involved in. Investigation of their interaction partners in a spatial and dynamic way is therefore of prime interest to understand their functions. This is however limited by the technical complexity of assessing it in endogenous conditions. A solution to close this gap is to determine protein interaction directly in the relevant tissues at endogenous expression levels using Forster resonance energy transfer (FRET). The ideal fluorophore pair for FRET must, however, fulfil specific requirements: (i) the emission and excitation spectra of the donor and acceptor, respectively, must overlap; (ii) they should not interfere with proper folding, activity, or localization of the fusion proteins; (iii) they should be sufficiently photostable in plant cells. Furthermore, the donor must yield sufficient photon counts at near-endogenous protein expression levels. Although many fluorescent proteins were reported to be suitable for FRET experiments, only a handful were already described for applications in plants. Herein, we compare a range of fluorophores, assess their usability to study RLK interactions by FRET-based fluorescence lifetime imaging (FLIM) and explore their differences in FRET efficiency. Our analysis will help to select the optimal fluorophore pair for diverse FRET applications.\n\nOne-sentence summaryWe compared the performances of several different fluorescent protein pairs to study membrane protein interaction in plants with FRET.

plant biology↗

bioRxiv2019

Cristae undergo continuous cycles of fusion and fission in a MICOS-dependent manner

The mitochondrial inner membrane can reshape under different physiological conditions. How and at which frequency this occurs in vivo and what are the molecular players involved is unknown. Here we show using state-of-the-art live-cell stimulated emission depletion (STED) super-resolution nanoscopy that crista junctions (CJs) are dynamically fusing and dividing in a reversible and balanced manner at a timescale of seconds. CJ dynamics is strongly reduced in the absence of the MICOS subunit MIC13. Staining of the cristae membrane using different protein markers or two inner mitochondrial membrane-specific dyes revealed that cristae also undergo continuous cycles of fusion and fission. These processes are dependent on MIC13 and occur at a timescale of seconds, resembling CJ dynamics. Our data further suggest that MIC60 acts as a docking platform pioneering CJ formation. Overall, by employing a variety of advanced imaging techniques including FRAP (Fluorescence-Recovery-After Photobleaching), SPT (Single-Particle-Tracking), live-cell STED and confocal Airyscan microscopy we demonstrate that cristae undergo continuous cycles of fusion and fission in a manner that is mechanistically linked to CJ formation and dynamics.

cell biology↗