Effective microwave-assisted approach to 1,2,3-triazolobenzodiazepinones via tandem Ugi reaction/catalyst-free intramolecular azide-alkyne cycloaddition.

A novel catalyst-free synthetic approach to 1,2,3-triazolobenzodiazepinones has been developed and optimized. The Ugi reaction of 2-azidobenzaldehyde, various amines, isocyanides, and acids followed by microwave-assisted intramolecular azide-alkyne cycloaddition (IAAC) gave a series of target heterocyclic compounds in moderate to excellent yields. Surprisingly, the normally required ruthenium-based catalysts were found to not affect the IAAC, only making isolation of the target compounds harder while the microwave-assisted catalyst-free conditions were effective for both terminal and non-terminal alkynes.

Tagging and Deleting of Endogenous Caveolar Components Using CRISPR/Cas9 Technology.

Here, we describe how to utilize CRISPR/Cas9 technology in the generation of tissue culture cells with fluorescently tagged caveolar components as well as cells deleted of endogenous caveolar components. As one example, we will describe tagging of EHD2, caveolar neck protein, with Green Fluorescent protein (eGFP) from endogenous loci (knock-in, KI). As another example, we will describe deletion (knock-out, KO) of Caveolin1 (Cav1), an essential caveolar component in NIH/3T3 cells.

Cyto-Mine: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing, and Monoclonality Assurance.

The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics.

Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.

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EHD Proteins Cooperate to Generate Caveolar Clusters and to Maintain Caveolae during Repeated Mechanical Stress.

Caveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. Here we make extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae.

Caveolae protect endothelial cells from membrane rupture during increased cardiac output.

Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions.

Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids.

Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels.

Applications of flow cytometry for measurement of autophagy.

Autophagy is a dynamic catabolic process that plays a major role in sequestering and recycling cellular components in multiple physiological and pathophysiological conditions. Despite recent progress in our understanding of the autophagic process there is still a shortage of robust methods for monitoring autophagy in live cells. Flow cytometry, a reliable and unbiased method for quantitative collection of data in a high-throughput manner, was recently utilized to monitor autophagic activity in live and fixed mammalian cells.

News from the caves: update on the structure and function of caveolae.

Recent data from the study of the cell biology of caveolae have provided insights both into how these flask-shaped invaginations of the plasma membrane are formed and how they may function in different contexts. This review discusses experiments that analyse the composition and ultrastructural distribution of protein complexes responsible for generating caveolae, that suggest functions for caveolae in response to mechanical stress or damage to the plasma membrane, that show that caveolae may have an important role during the signalling events for regulation of metabolism, and that imply that caveolae can act as endocytic vesicles at the plasma membrane. We also highlight unexpected roles for caveolar proteins in regulating circadian rhythms and new insights into the way in which caveolae may be involved in fatty acid uptake in the intestine.

Deletion of cavin genes reveals tissue-specific mechanisms for morphogenesis of endothelial caveolae.

Caveolae are abundant in endothelial cells and are thought to have important roles in endothelial cell biology. The cavin proteins are key components of caveolae, and are expressed at varied amounts in different tissues. Here we use knockout mice to determine the roles of cavins 2 and 3 in caveolar morphogenesis in vivo.

Biogenesis and cargo selectivity of autophagosomes.

Autophagy is a major catabolic pathway in eukaryotes, which is required for the lysosomal/vacuolar degradation of cytoplasmic proteins and organelles. Interest in the autophagy pathway has recently gained momentum largely owing to identification of multiple autophagy-related genes and recognition of its involvement in various physiological conditions. Here we review current knowledge of the molecular mechanisms regulating autophagy in mammals and yeast, specifically the biogenesis of autophagosomes and the selectivity of their cargo recruitment.

LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis.

Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion.

Dissecting the involvement of LC3B and GATE-16 in p62 recruitment into autophagosomes.

Autophagy is a major intracellular trafficking pathway that delivers proteins and organelles from the cytoplasm into lysosomes for consequential degradation and recycling. Mammalian Atg8s are key autophagic factors that undergo a unique ubiquitin-like conjugation to the lipid phase of the autophagosomal membrane. In addition to their activity in autophagosome formation, several Atg8s directly bind p62/SQSTM1.

LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis.

Autophagy, a critical process for bulk degradation of proteins and organelles, requires conjugation of Atg8 proteins to phosphatidylethanolamine on the autophagic membrane. At least eight different Atg8 orthologs belonging to two subfamilies (LC3 and GATE-16/GABARAP) occur in mammalian cells, but their individual roles and modes of action are largely unknown. In this study, we dissect the activity of each subfamily and show that both are indispensable for the autophagic process in mammalian cells.

Lipophagy: selective catabolism designed for lipids.

Until recently, degradation of lipid droplets (LDs) has been thought to take place in the cytosol by resident lipases. In a recent issue of Nature, Singh and coworkers describe the involvement of selective autophagy in the delivery of lipid droplets for lysosomal degradation.

A role for NBR1 in autophagosomal degradation of ubiquitinated substrates.

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains.

Flow cytometric analysis of autophagy in living mammalian cells.

Autophagy is a major intracellular catabolic pathway induced in response to amino acid starvation. Recent findings implicate it in diverse physiological/pathophysiological events, such as protein and organelle turnover, development, aging, pathogen infection, cell death, and neurodegeneration. However, experimental methods to monitor this process in mammalian cells are limited because of the deficiency of autophagic markers.

Autophagy-independent incorporation of GFP-LC3 into protein aggregates is dependent on its interaction with p62/SQSTM1.

LC3 is a widely used marker of autophagosomes in mammalian cells. However, in addition to its autophagosomal localization, GFP-LC3 is often found associated with protein aggregates that are formed in an autophagy-independent manner. In addition, LC3 directly interacts with p62/SQSTM1 (hereafter named p62), a common constituent of protein aggregates.

The N-terminus and Phe52 residue of LC3 recruit p62/SQSTM1 into autophagosomes.

LC3 belongs to a novel ubiquitin-like protein family that is involved in different intracellular trafficking processes, including autophagy. All members of this family share a unique three-dimensional structure composed of a C-terminal ubiquitin core and two N-terminal alpha-helices. Here, we focus on the specific contribution of these regions to autophagy induced by amino acid deprivation.

Utilizing flow cytometry to monitor autophagy in living mammalian cells.

Autophagy is a major intracellular catabolic pathway that takes part in diverse biological events including response to amino acid starvation, protein and organelle turnover, development, aging, pathogen infection and cell death. However, experimental methods to monitor this process in mammalian cells are limited due to lack of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, was shown to selectively incorporate into autophagosome, thus serving as a unique bona fide marker of autophagosomes in mammals.

Oxidation as a post-translational modification that regulates autophagy.

The toxicity associated with accumulation of reactive oxygen species (ROS) has led to the evolution of various defense strategies to overcome oxidative stress, including autophagy. This pathway is involved in the removal and degradation of damaged mitochondria and oxidized proteins. At low levels, however, ROS act as signal transducers in various intracellular pathways.

Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.

Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles. This pathway is activated under environmental stress conditions, during development and in various pathological situations. In this study, we describe the role of reactive oxygen species (ROS) as signaling molecules in starvation-induced autophagy.

Microtubules support production of starvation-induced autophagosomes but not their targeting and fusion with lysosomes.

Autophagy is a major catabolic pathway in eukaryotic cells whereby the lack of amino acids induces the formation of autophagosomes, double-bilayer membrane vesicles that mediate delivery of cytosolic proteins and organelles for lysosomal degradation. The biogenesis and turnover of autophagosomes in mammalian cells as well as the molecular mechanisms underlying induction of autophagy and trafficking of these vesicles are poorly understood. Here we utilized different autophagic markers to determine the involvement of microtubules in the autophagic process.