Mitochondria: Practical Protocols

ISBN-13: 9781627038331 / Angielski / Miękka / 2014 / 586 str.

Dario Leister;Johannes Herrmann

Opis

Mitochondrial Genomics and Proteomics Protocols offers a broad collection of methods for studying the molecular biology, function, and features of mitochondria. In the past decade, mitochondrial research has elucidated the important influence of mitochondrial processes on integral cell processes such as apoptosis and cellular aging. This practical guide presents a wide spectrum of mitochondrial methods, each written by specialists with solid experience and intended for implementation by novice and expert researchers alike.
Part I introduces major experimental model systems and discusses their specific advantages and limitations for functional analysis of mitochondria. The concise overview of general properties of mitochondrial systems is supplemented by detailed protocols for cultivation of model organisms. Parts II-VI comprise a robust collection of protocols for studying different molecular aspects of mitochondrial functions including: genetics and microbiology, biochemistry, physiology, dynamics and morphology, and functional genomics. Emphasis is placed on new and emerging topics in mitochondrial study, such as the examination of apoptotic effects, fusion and fission of mitochondria, and proteome and transcriptome analysis.

Spis treści

Chapter Title 0 Preface Section 1: Model systems to study mitochondrial functions 1 The Mitochondria of Cultured Mammalian Cells. I: Analysis by Immunofluorescence Microscopy, Histochemistry, Subcellular Fractionation and Cell Fusion 2 The Mitochondria of Cultured Mammalian Cells. II: Expression and visualization of exogenous proteins in fixed and live cells 3 Drosophila melanogaster as a Model System to Study Mitochondrial Function 4 Isolation and Functional Analysis of Mitochondria from the Nematode Caenorhabditis elegans 5 Isolation of Mitochondria from Procyclic Trypanosoma brucei 6 Saccharomyces cerevisiae as a Model Organism to Study Mitochondrial Biology - General Considerations and Basic Procedures 7 Studying mitochondria in an attractive model: Schizosaccharomyces pombe 8 Neurospora crassa as a Model Organism for Mitochondrial Biogenesis 9 Isolation of intact, functional mitochondria from the model plant, Arabidopsis thaliana 10 Chlamydomonas reinhardtii: the model of choice to study mitochondria from unicellular photosynthetic organisms Section 2: Genetics and Molecular Biology 11 Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination 12 Screens for Mitochondrial Mutants in Yeast 13 Transcript end mapping and analysis of RNA editing in plant mitochondria 14 In Vitro Analysis of the Yeast Mitochondrial RNA Polymerase 15 Functional Analysis by Inducible RNA Interference in Drosophila melanogaster 16 Analysis of Replicating Mitochondrial DNA by Two Dimensional Agarose Gel Electrophoresis Section 3: Biochemistry 17 The Analysis of tRNA Import into Mammalian Mitochondria 18 Analysis of mitochondrial protein synthesis in yeast 19 Large Scale Isolation of Mitochondrial Ribosomes from Mammalian Tissues 20 Protein Import into Isolated Mitochondria 21 Analyzing import intermediates of mitochondrial proteins by blue native gel electrophoresis 22 In vitro and in vivo Protein Import into Plant Mitochondria 23 Two dimensional blue native / blue native polyacrylamide gel electrophoresis for the characterization of mitochondrial protein complexes and supercomplexes 24 Investigation of Iron-Sulfur Protein Maturation in Eukaryotes 25 Studying Proteolysis within Mitochondria Section 4: Physiology 26 Methods to Determine the Status of Mitochondrial ATP Synthase Assembly 27 ATP production in Isolated Mitochondria of Procyclic Trypanosoma brucei 28 Oxidative Stress and Plant Mitochondria 29 Measuring mitochondrial shape changes and their consequences on the mitochondrial involvement during apoptosis 30 Assessing mitochondrial potential, calcium, and redox state in isolated mammalian cells using confocal microscopy Section 5: Dynamics and Morphology 31 Fluorescence imaging of mitochondria in yeast 32 Mitochondrial Fusion in vitro 33 Conventional and Immunoelectron Microscopy of Mitochondria 34 Mitochondrial division in C. elegans Section 6: Functional Genomics 35 Yeast Mitochondrial Transcriptomics 36 Plant Mitochondrial Transcriptomics by Quantitative Real-Time PCR 37 Proteomics of Yeast Mitochondria 38 Arabidopsis Mitochondrial Proteomics 39 MitoP2, an integrated database for mitochondrial proteins