From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
1Life Sciences Division, Lawrence Berkeley National Laboratory, 2Joint Bioenergy Institute, Physical Biosciences Division, Lawrence Berkeley National Laboratory, 3National Energy Research Scientific Computing Center, Lawrence Berkeley National Laboratory
The bottleneck for cellular 3D electron microscopy is feature extraction (segmentation) in highly complex 3D density maps. We have developed a set of criteria, which provides guidance regarding which segmentation approach (manual, semi-automated, or automated) is best suited for different data types, thus providing a starting point for effective segmentation.
Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture
1Max von Pettenkofer Institute, 2Department of Medicine, University of Cambridge, 3Institute for Informatics, Ludwig-Maximilians-University Munich
Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.
Published August 8, 2013. Keywords: Genetics, Cellular Biology, Molecular Biology, Microbiology, Biochemistry, Eukaryota, Investigative Techniques, Biological Phenomena, Gene expression profiling, RNA synthesis, RNA processing, RNA decay, 4-thiouridine, 4sU-tagging, microarray analysis, RNA-seq, RNA, DNA, PCR, sequencing
1Department of Biology, University of Alabama at Birmingham, 2Nutrition Métabolisme Aquaculture, INRA UR1067, 3Laboratoire de Physiologie et Genomique des Poissons, INRA UR1037
In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.
1Unité de Génomique Virale et Vaccination, Virology Department, Institut Pasteur, CNRS UMR3569, 2Unité de Chimie et Biocatalyse, Biochemistry and Structural Biology Department, Institut Pasteur, CNRS UMR3523, 3Unité des Interactions Moléculaires Flavivirus-Hôtes, Virology Department, Institut Pasteur
In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.
Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas
1Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 2Department of Chemical and Biomolecular Engineering, University of Tennessee
Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.
Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.
Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
1Department of Life Sciences, National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev
To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.
Published December 18, 2013. Keywords: Biochemistry, aging, Caenorhabditis elegans, heat shock response, neurodegenerative diseases, protein folding homeostasis, proteostasis, stress, temperature-sensitive
1Department of Structural Biology, Max Planck Institute of Biophysics
We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.
Published September 14, 2014. Keywords: Structural Biology, electron microscopy, electron cryo-tomography, mitochondria, ultrastructure, membrane structure, membrane protein complexes, ATP synthase, energy conversion, bioenergetics
Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining
1Virus and Centromere Team, Centre de Génétique et Physiologie Moléculaire et Cellulaire, CNRS UMR 5534, 2Université de Lyon 1, 3Laboratoire d'excellence, LabEX DEVweCAN, 4Institut de Virologie Moléculaire et Structurale, CNRS UPR 3296, 5Centre de Recherche en Cancérologie de Lyon, INSERM U1052, CNRS UMR 5286
We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.