Fast and Long-Term Super-Resolution Imaging of Endoplasmic Reticulum 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. Herein, these challenges are addressed by using ultra-low irradiation intensities and a neural network for image restoration, enabling extensive imaging of single living cells. The endoplasmic reticulum (ER) is chosen as the target structure due to its dynamic nature over short and long timescales. The reduced irradiation intensity combined with denoising permits continuous ER dynamics observation in living cells for up to 7 h with a temporal resolution of seconds. This allows 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 ultralow irradiation with image restoration enables comprehensive analysis of organelle dynamics over extended periods in living cells

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands

Synergizing exchangeable fluorophore sabels for multitarget STED microscopy

Investigating the interplay of cellular proteins with optical microscopy requires multitarget 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. Noncovalent, 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 six-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching as well as facilitate long acquisition times and multicolor live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increases the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images

Synergizing exchangeable fluorophore labels for multi-target STED microscopy

AbstractInvestigating 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.</jats:p