Biology subjects

Barth, H.-D.

Publications and source records attributed to Barth, H.-D..

3 recordsLinked to original sources

bioRxiv2024

Fast and long-term super-resolution imaging of ER nano-structural dynamics in living cells using a neural network

Stimulated emission depletion (STED) microscopy is a super-resolution technique that surpasses the diffraction limit and has contributed to the study of dynamic processes in living cells. However, high laser intensities induce fluorophore photobleaching and sample phototoxicity, limiting the number of fluorescence images obtainable from a living cell. Here, we address these challenges by using ultra-low irradiation intensities and a neural network for image restoration, enabling extensive imaging of single living cells. The endoplasmic reticulum (ER) was chosen as the target structure due to its dynamic nature over short and long timescales. The reduced irradiation intensity combined with denoising permitted continuous ER dynamics observation in living cells for up to 7 hours with a temporal resolution of seconds. This allowed for quantitative analysis of ER structural features over short (seconds) and long (hours) timescales within the same cell, and enabled fast 3D live-cell STED microscopy. Overall, the combination of ultra-low irradiation with image restoration enables comprehensive analysis of organelle dynamics over extended periods in living cells.

biophysics↗

bioRxiv2023

Single-molecule FRET and molecular dynamics simulations reveal early activation steps of MET receptor by Listeria monocytogenes

The assembly of membrane receptors into signaling complexes is at the origin of key cellular events. Yet, we often lack detailed structural mechanistic understanding. Receptors are embedded into a complex cellular membrane, which defines their dynamics but also complicates their experimental characterizations significantly. Here, we showcase an integrative structural biology approach to investigate the activation mechanism of the human growth factor receptor MET. MET is a receptor tyrosine kinase involved in cell proliferation, migration, and survival. MET is also hijacked by the intracellular pathogen Listeria monocytogenes. Its invasion protein, internalin B (InlB), binds to MET and promotes the formation of a signaling dimer that triggers the internalization of the pathogen. Crystallography had suggested two different 2:2 MET:InlB complexes. Here, we use a combination of structural biology, modeling, molecular dynamics simulations, and in situ single-molecule Forster resonance energy transfer (smFRET) to elucidate the early events in MET activation. Simulations show that InlB binding stabilizes MET in a conformation that promotes dimer formation. smFRET identifies the organization of the in situ signaling dimer, which resembles one of the two crystal structures yet shows differences. Further MD simulations resulted in a refinement of the dimer model, which is in quantitative agreement with smFRET results. We accurately describe the structural dynamics underpinning an important cellular event and introduce a powerful methodological pipeline applicable to studying the activation of other plasma membrane receptors in situ.

biophysics↗

bioRxiv2022

Synergizing exchangeable fluorophore labels for multi-target STED microscopy

Investigating the interplay of cellular proteins with optical microscopy requires multi-target labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging experiment. Advanced microscopy methods such as STED microscopy additionally demand balanced excitation, depletion and emission wavelengths for all fluorophores, further reducing multiplexing capabilities. Non-covalent, weak-affinity labels bypass this "spectral barrier" through label exchange and sequential imaging of different targets. Here, we combine exchangeable HaloTag ligands, weak-affinity DNA hybridization and hydrophophic and protein-peptide interactions to increase labeling flexibility and demonstrate 6-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching, facilitate long acquisition times and multi-color live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increase the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images.

biophysics↗