A biocompatible supramolecular hydrogel mesh for sample stabilization in light microscopy and nanoscopy.

Most embedding media for live and fixed samples were not designed for microscopy and have issues including long polymerization times, peak of toxicity toward the sample during the sol-gel transition, and irreversibility of this transition. Gels derived from biological sources are widely used in microscopy, but their precise composition is ill-defined and can vary between batches. Non-physiological temperatures and/or specific enzymatic solutions are often needed to revert the gel back to the sol state to allow sample recovery.

Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis.

How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over two hours. While a significant fraction of Condensins remains chromatin-bound until early G1, Cohesin-STAG1 and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes and are sufficient to build the first interphase TAD structures.

The reproduction of gram-negative protoplasts and the influence of environmental conditions on this process.

Bacterial protoplasts are known to reproduce independently of canonical molecular biological processes. Although their reproduction is thought to be influenced by environmental conditions, the growth of protoplasts in their natural habitat has never been empirically studied. Here, we studied the life cycle of protoplasts in their native environment.

Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging.

Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer.

Protocol for the setup and use of a human choroid plexus endothelial-epithelial two-cell-type model.

Choroid plexus, located in brain ventricles, is the site of blood-cerebrospinal fluid barrier that contains endothelial cells and an epithelial monolayer separated by stroma. We established a two-cell-type model of the human choroid plexus consisting of immortalized endothelial cells (iHCPEnC) and epithelial papilloma (HIBCPP) cells grown on opposite sides of filter supports. In this protocol, we describe the preparation of this model, the measurement of transepithelial electrical resistance (TEER), and immunofluorescence imaging-based analysis to determine the barrier function.

Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates.

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain.

Immortalized human choroid plexus endothelial cells enable an advanced endothelial-epithelial two-cell type model of the choroid plexus.

The choroid plexus (CP) is a highly vascularized structure containing endothelial and epithelial cells located in the ventricular system of the central nervous system (CNS). The role of the fenestrated CP endothelium is under-researched and requires the generation of an immortalized CP endothelial cell line with preserved features. Transduction of primary human CP endothelial cells (HCPEnC) with the human telomerase reverse transcriptase (hTERT) resulted in immortalized HCPEnC (iHCPEnC), which grew as monolayer with contact inhibition, formed capillary-like tubes in Matrigel, and showed no colony growth in soft agar.

Liquid-to-solid phase transition of oskar ribonucleoprotein granules is essential for their function in Drosophila embryonic development.

Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition.

Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum.

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body.

The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction.

Neurons have a membrane periodic skeleton (MPS) composed of actin rings interconnected by spectrin. Here, combining chemical and genetic gain- and loss-of-function assays, we show that in rat hippocampal neurons the MPS is an actomyosin network that controls axonal expansion and contraction. Using super-resolution microscopy, we analyzed the localization of axonal non-muscle myosin II (NMII).

Stabilization of chromatin topology safeguards genome integrity.

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex. How this pathway reflects and influences the three-dimensional nuclear architecture is not known.

Protein-Specific, Multicolor and 3D STED Imaging in Cells with DNA-Labeled Antibodies.

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir.

Photoactivation of silicon rhodamines via a light-induced protonation.

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released.

Independent yet overlapping pathways ensure the robustness and responsiveness of trans-Golgi network functions in .

The trans-Golgi-network (TGN) has essential housekeeping functions in secretion, endocytosis and protein sorting, but also more specialized functions in plant development. How the robustness of basal TGN function is ensured while specialized functions are differentially regulated is poorly understood. Here, we investigate two key regulators of TGN structure and function, ECHIDNA and the Transport Protein Particle II (TRAPPII) tethering complex.

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion.

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described.

Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins: The Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells.

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells.

Ultrastructural Characterization of Zika Virus Replication Factories.

A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles.

Clathrin coated pits, plaques and adhesion.

Clathrin mediated endocytosis (CME) is the main route of receptor internalization in mammalian cells and this well conserved mechanism has been intensively studied for over 40yrs. In the general or 'canonical' model of CME clathrin coated pits form stochastically at the plasma membrane and coated pit curvature develops as the coated pit grows through clathrin polymerization. However, the canonical model of CME does not explain the diversity of endocytically active clathrin coated structures (CCSs) found at the plasma membrane by both electron and light microscopy.

Essential Role of the PfRh5/PfRipr/CyRPA Complex during Plasmodium falciparum Invasion of Erythrocytes.

Plasmodium falciparum parasites in the merozoite stage invade human erythrocytes and cause malaria. Invasion requires multiple interactions between merozoite ligands and erythrocyte receptors. P.

Detailed morphological characterisation of Hendra virus infection of different cell types using super-resolution and conventional imaging.

Background: Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.

Combining wide-field super-resolution microscopy and electron tomography: rendering nanoscopic correlative arrays on subcellular architecture.

In this chapter, the authors outline in full detail, an uncomplicated approach that enables the combination of wide-field fluorescence super-resolution microscopy with electron tomography, thereby providing an approach that affords the best possible confidence in the structures investigated. The methodical steps to obtain these high-throughput correlative nanoscopic arrays will be visually explored and outlined in detail. The authors will demonstrate the feasibility of the method on cultured Caco-2 colorectal cancer cells that are labeled for filamentous actin.