Biology subjects

Köhler, R.

Publications and source records attributed to Köhler, R..

6 recordsLinked to original sources

bioRxiv2026

Structural and functional heterogeneity in cardiac RyR signalling nanodomains revealed with quantitative single molecule mapping toolkit

The nanoscale organisation of ryanodine receptor type 2 (RyR2) channels and junctophilin-2 (JPH2) shapes cardiac Ca{superscript 2} release, but how this relationship is remodelled in right ventricular failure remains unclear. We developed an integrated analysis pipeline building on multiplexed DNA-PAINT data to quantify RyR2 and JPH2 abundance, stoichiometry of co-clustering, and spatial organisation within individual subsarcolemmal Ca{superscript 2}-release nanodomains. Applied to cardiomyocytes from control rats with pulmonary hypertension-induced right ventricular failure, the approach revealed reduced co-localisation between RyR2 and JPH2 within peripheral junctions and greater variability in their co-clustering stoichiometry across the cell. A sub-variogram analysis further showed divergent remodelling of JPH2 expression patterns across subcellular length scales, indicating that disease alters both local molecular composition and cell-wide spatial heterogeneity. Experimentally derived RyR2/JPH2 maps were then used to model as two-dimensional templates for stochastic reaction-diffusion of Ca{superscript 2} release. Simulations of spontaneous Ca{superscript 2} waves from failing cells showed up to 25% wave propagation. Maps from failing cells supported faster transverse and longitudinal Ca{superscript 2} dependent release, consistent with the emergence, that support the likelihood of heterogeneous modulation and coupling of RyR resulting from the RyR redistribution and heterogeneous JPH2 expression in the failing cell. Together, these findings identify spatially heterogeneous RyR2-JPH2 remodelling as a potential substrate for dysregulated Ca{superscript 2} signalling in right ventricular failure and establish a transferable toolkit for linking molecular nanostructure to cellular function.

cell biology↗

bioRxiv2023

Putting the Par back into ParABS: Plasmid Partitioning Driven by ParA Oscillations

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy number plasmids. However, despite extensive research, the spatio-temporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo has remained unclear. In this study, we utilise high-throughput imaging, quantitative data analysis, and computational modelling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ( flips) between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behaviour of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

microbiology↗

bioRxiv2022

Dynamic bimodality of curli expression in planktonic cultures of Escherichia coli is stabilized by cyclic-di-GMP regulation

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli expression occurs not only in submerged and macrocolony biofilms, but also in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells under all tested growth conditions. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of c-di-GMP regulation. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

microbiology↗

bioRxiv2022

High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS

The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB a protein that binds to centromeric-like parS sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We identify ParA hopping on the nucleoid as the primary determinant of this geometry-sensing and develop a unified computational model that quantitatively explains the observed behaviour. Furthermore, we confirm our model prediction that cells with the lowest plasmid concentration transition to oscillatory dynamics and find similar behaviour in a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, minimising ATP consumption. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.

biophysics↗

bioRxiv2019

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

Here we describe tissue-resident memory B lymphocytes of spleen and bone marrow. Single cell transcriptomes and B cell receptor repertoires identify several exclusive populations of isotype-switched memory B cells (Bsm) in murine spleen and bone marrow, and one interconnected population of 10-20%. A population of marginal zone-like Bsm is located exclusively in the spleen while a novel population of quiescent Bsm is located exclusively in the bone marrow. Cells of two further populations, present in both, spleen and bone marrow, differ in repertoire between the two organs, i.e. are resident as well. Finally, another interconnected population of Bsm of the B1 lineage is present in spleen and bone marrow. In the bone marrow, all Bsm individually dock onto VCAM1+ stromal cells, resting in terms of activation, proliferation and mobility. The discrete B cell memory of bone marrow may be key to rapid secondary humoral responses to systemic antigens.

immunology↗

bioRxiv2019

KCa3.1-transgene induction in murine skin produces pruritic eczematous dermatitis with severe epidermal hyperplasia and hyperkeratosis

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-{beta}1 (60-fold), IL-23 (34-fold), IL-6 (33-fold), and TNF (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-{beta}1 (-88%), IL-23 (-77%), and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.

physiology↗