Automated STED nanoscopy for high-throughput imaging of cellular structures

STimulated Emission Depletion (STED) nanoscopy uniquely combines a high spatial resolution (20-50nm in cells) with relatively fast imaging (frame rate of ∼1-30Hz), straightforward sample preparation and direct image output (no postprocessing required). Although these characteristics in principle make STED very suitable for high-throughput imaging, only few steps towards automation have been made. Here, we have developed fully automated STED imaging, eliminating all manual steps including the selection and characterisation of the relevant (cellular) regions, sample focusing and positioning, and microscope adjustments. This automatic STED image acquisition increases the data output by roughly two orders of magnitude, resulting in a more efficient use of the high-end microscope, and the ability to detect and characterise objects that are only present in a small subset of the sample

Fluorescence-based super-resolution-microscopy strategies for chromatin studies

Super-resolution microscopy (SRM) is a prime tool to study chromatin organisation at near biomolecular resolution in the native cellular environment. With fluorescent labels DNA, chromatin-associated proteins and specific epigenetic states can be identified with high molecular specificity. The aim of this review is to introduce the field of diffraction-unlimited SRM to enable an informed selection of the most suitable SRM method for a specific chromatin-related research question. We will explain both diffraction-unlimited approaches (coordinate-targeted and stochastic-localisation-based) and list their characteristic spatio-temporal resolutions, live-cell compatibility, image-processing, and ability for multi-colour imaging. As the increase in resolution, compared to, e.g. confocal microscopy, leads to a central role of the sample quality, important considerations for sample preparation and concrete examples of labelling strategies applicable to chromatin research are discussed. To illustrate how SRM-based methods can significantly improve our understanding of chromatin functioning, and to serve as an inspiring starting point for future work, we conclude with examples of recent applications of SRM in chromatin research.</p

Chemogenetic Tags with Probe Exchange for Live-Cell Fluorescence Microscopy

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions

STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA-adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodeling. </p

Mouse Heterochromatin Adopts Digital Compaction States without Showing Hallmarks of HP1-Driven Liquid-Liquid Phase Separation

Mouse cells package heterochromatin into compact foci. Erdel et al. show that these foci lack hallmarks of liquid droplets and rather resemble collapsed polymer globules. Their size, accessibility, and compaction are independent of HP1. They can adopt two distinct folding states that possibly represent the fundamental modes of chromatin compaction

Ring-colocalization:Analysis of dual-color images of ring-like structures.

Analysis of dual-color images of ring-like structures. Code to determine the relative positions of two ring-like structures (no circle-fitting involved). This code has been implemented to determine the relative positions of ESCRT-III proteins during cell division in the archaea Sulfolobus acidocaldarius: Hurtig, F., Burgers, T.C.Q., Cezanne, A., Jiang, X., Mol, F.N., Traparić, J., Tarrason-Risa, G., Pulschen, A.A., Harker-Kirschneck, L, Šarić, A., Vlijm, R. & Baum, B. The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division. BioRxiv (2022) https://doi.org:10.1101/2022.09.16.508273 Script developed by: Frank N. Mol & Rifka Vlijm Molecular Biophysics Zernike Institute of Advanced Materials University of Groninge

Super-Resolution Imaging of Peroxisomal Proteins Using STED Nanoscopy

Peroxisomes are crucial organelles that occur in almost all eukaryotes. Well known are their roles in various metabolic processes, such as hydrogen peroxide detoxification and lipid metabolism. Recent studies indicated that peroxisomes also have several non-metabolic functions, for instance, in stress response, signaling, and cellular ageing. In mammalian cells, the small size of peroxisomes (~200 nm, near the diffraction limit) hinders unveiling peroxisomal structures by conventional light microscopy. However, in the yeast Hansenula polymorpha, they can reach up to 1.5 μm in diameter, depending on the carbon source. To study the localization of peroxisomal proteins in cells in more detail, super-resolution imaging techniques such as stimulated emission depletion (STED) microscopy can be used. STED enables fast (live-cell) imaging well beyond the diffraction limit of light (30-40 nm in cells), without further data processing. Here, we present optimized protocols for the fluorescent labeling of specific peroxisomal proteins in fixed and living cells. Moreover, detailed measurement protocols for successful STED imaging of human and yeast peroxisomes (using antibodies or genetic tags labeled with dyes) are described, extended with suggestions for individual optimizations.</p

Super-Resolution Imaging of Peroxisomal Proteins Using STED Nanoscopy

Peroxisomes are crucial organelles that occur in almost all eukaryotes. Well known are their roles in various metabolic processes, such as hydrogen peroxide detoxification and lipid metabolism. Recent studies indicated that peroxisomes also have several non-metabolic functions, for instance, in stress response, signaling, and cellular ageing. In mammalian cells, the small size of peroxisomes (~200 nm, near the diffraction limit) hinders unveiling peroxisomal structures by conventional light microscopy. However, in the yeast Hansenula polymorpha, they can reach up to 1.5 μm in diameter, depending on the carbon source. To study the localization of peroxisomal proteins in cells in more detail, super-resolution imaging techniques such as stimulated emission depletion (STED) microscopy can be used. STED enables fast (live-cell) imaging well beyond the diffraction limit of light (30-40 nm in cells), without further data processing. Here, we present optimized protocols for the fluorescent labeling of specific peroxisomal proteins in fixed and living cells. Moreover, detailed measurement protocols for successful STED imaging of human and yeast peroxisomes (using antibodies or genetic tags labeled with dyes) are described, extended with suggestions for individual optimizations