Cover: In Nazari et al. (http://doi.org/10.1002/cpcb.105), the image shows spheroids with encapsulating basement membrane in 3D collagen gels. (A) MCF10A spheroids confined by a basement membrane stained with collagen IV antibody and imaged using a confocal microscope. (B) MDA-231BO spheroid stained for collagen IV is shown as a confocal slice and a max-projection of the collagen IV surrounding the 3D spheroid. (C) High-resolution images of MDA-231BO spheroids with an intact basement membrane before and after initial break through the basement membrane. (D) Six days after spheroids were incubated in 3D collagen gels, cells migrated into the 3D ECM. Note that the absence of a basement membrane or collagen IV staining is because the cells have broken out and migrated away from the initial spheroid, which is depicted by dashed lines.

Cover: In Rezanejad et al. (https://doi.org/10.1002/cpcb.82). Intact whole-mount immunostaining of organoids after growth for 14 days. See e82.

About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing. Here, we describe a FACS-based method to select populations of live mutant cells based on the surface levels of endogenous proteins or engineered reporters. Collection of these mutant populations enables subsequent deep sequencing and bioinformatics analysis to identify genes that regulate the trafficking pathway. This method can be readily adapted to genetically dissect a broad range of mammalian membrane trafficking processes using haploid genetics or CRISPR-Cas9 screens. © 2018 by John Wiley & Sons, Inc.

Many cells possess the ability to repair plasma membrane disruption in physiological conditions. Growing evidence indicates a correlation between membrane repair and many human diseases. For example, a negative correlation is observed in muscle where failure to reseal sarcolemma may contribute to the development of muscular dystrophies. Instead, a positive correlation is observed in cancer cells where membrane repair may be exacerbated during metastasis. Here we describe a protocol that combines laser technology for membrane damage, immunostaining with gold nanoparticles and imaging by fluorescence microscopy and transmission electron microscopy (TEM), which allows the characterization of the molecular machinery involved in membrane repair. Fluorescence microscopy enables to determine the subcellular localization of candidate proteins in damaged cells while TEM offers high-resolution ultrastructural analysis of the µm²-disruption site, which enables to decipher the membrane repair mechanism. Here we focus on the study of human skeletal muscle cells, for obvious clinical interest, but this protocol is also suitable for other cell types. © 2018 by John Wiley & Sons, Inc.

To date, the permeability of epithelia to larger solutes (greater than ∼4 Å in diameter) has been analyzed by flux measurements using various tracers that cannot spatially resolve the permeation sites. This unit describes a method for localizing such sites of passage in epithelial sheets with subcellular resolution. The method makes use of avidin as a basolateral capture probe in epithelial monolayers or mucosae to unmask the passage of biotinylated and fluorophore-labeled tracer molecules as they go through the junctional barrier. Once bound to avidin, the tracers are immobilized at the site of a barrier leak. The localization, the distribution, and the extent of passage are eventually evaluated by imaging. The assay detects single leaks and is hence able to spatially resolve rarely occurring changes. It is also modular and flexible to use with various macromolecular tracers, and its sensitivity is adjustable. If designed as a chase experiment, the method allows for analysis of temporal barrier openings. If performed at low temperatures, this assay will block transcellular passage and, combined with global flux measurement, unambiguously determine paracellular passage. © 2018 by John Wiley & Sons, Inc.

The molecular interactions and translocation of signal transduction factors in individual cells can be imaged by fluorescence microscopy. Alternatively, downstream promoter activity in single cells can be imaged by bioluminescence microscopy. However, the same stimuli can lead to different gene expression responses in individual cells. For this reason, it is desirable to simultaneously image signal transduction and gene expression events in the same cells. Here, we describe a method that combines fluorescence and bioluminescence microscopy to image protein kinase C (PKC) translocation from the cytosol to the plasma membrane and the expression of nuclear factor kappa-light polypeptide B (NF-κB)-regulated genes. © 2017 by John Wiley & Sons, Inc.

G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.

Chemotaxis and cell migration play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Novel chemotaxis/migration inhibitors are desirable for developing effective therapeutics and probing molecular mechanisms. We describe a fluorescence-based phenotypic assay in a 1536-well plate format for high-throughput screening of novel inhibitors of chemotaxis/migration within complex libraries of thousands of compounds. Although the assay utilizes the unique cellular response properties of Dictyostelium, the compounds identified are able to inhibit chemotaxis of mammalian cells. In addition, a parallel cell cytotoxicity counter-screen with an ATP content assay is described that eliminates cytotoxic compounds from the screen. This novel compound screening approach enables rapid identification of novel lead compounds that inhibit chemotaxis in human and other cells for drug development and research tools. © 2017 by John Wiley & Sons, Inc.

Complex interaction networks orchestrate key cellular processes including but not limited to transcription, translation, metabolism, and cell signaling. Delineating these interactions will aid in deciphering the regulation and function of these pathways and potential for manipulation. Proximity-dependent biotin identification (BioID) is quickly gaining popularity as a powerful tool for identifying novel protein-protein and proximity-based interactions in live cells. This technique relies on a promiscuous biotin ligase, which is fused to a protein of interest and, upon expression in the desired cell, will biotinylate proximal endogenous proteins. In vivo protein-protein interactions can be very transient and occur momentarily to facilitate signaling or a metabolic function. BioID is useful in identifying these weak and/or transient interactions that are not detected by traditional methods such as yeast two-hybrid or affinity purification. Here, we outline a BioID protocol that can be used as a workflow to guide a new application. © 2016 by John Wiley & Sons, Inc.

Isolation of murine liver sinusoidal endothelial cells (LSECs) is an exacting and finicky procedure. After exhaustive standardization, we were able to devise an easily reproducible protocol which produced consistent results. Moreover, we scripted a protocol which clarifies even the smallest of steps, following which isolation of LSECs is made significantly easier. Using the standardized LSEC isolation protocol herein, we demonstrated that the bacterial toxin pyocyanin (from Pseudomonas aeruginosa) induced a significant dose-dependent reduction in LSEC porosity, this being preventable by the enzyme catalase, but not by the enzyme superoxide dismutase.