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Essential cell biology. v. 2, cell function : a practical approach / edited by John Davey and Mike Lord. — Oxford ;New York : Oxford University Press, 2003.—(58.15/E78/v.2)

Contents

        Contents
    
    List of protocols page xi
    Abbreviations xv
    1 Enzyme activities 1
    Keith F. Tipton
    1 Introduction 1
    2 Enzyme specificity and nomenclature 1
    3 Determination of enzyme activity 1
    Reaction progress curves 2
    The effects of enzyme concentration and expression of activities 3
    Conditions for activity measurements 4
    4 The effects ofsubstrate concentration 5
    The Michaelis-Menten relationship 5
    Failure to obey the Michaelis-Menten equation 5
    5 Enzyme inhibitors 9
    Reversible inhibitors 9
    Tight-binding inhibitors 26
    Irreversible inhibitors 29
    6 Behaviour in vivo 40
    Recovery from irreversible inhibition 40
    Effects of cellular processes 41
    Inhibitor specificity 43
    References 44
    2 Gene expression 47
    Luis Parada, Cem Elbi, Miroslav Dundr, and Tom Misteli
    1 Introduction 47
    2 Indirect immunofluorescence 48
    3 GFP in fixed or living cells 51
    4 Nuclear matrix preparation 53
    5 Visualization of transcription sites 55
    6 Fluorescence in situ hybridization 59
    7 Chromosome preparation and staining 67
    8 Fluorescent chromosome painting 70
    References 76
    3 Regulation of the cell cycle 79
    Diana M. Gitig and Andrew Koff
    1 Introduction 79
    2 Cell biology: detection of cell cycle arrests 79
    3 Genetics: knockout and transgenic mice 83
    4 Biochemistry 85
    In vitro: recombinant proteins 85
    In viv0: extract reconstitution assays 86
    5 Conclusions 91
    Acknowledgements 91
    References 91
    4 Cellular bioenergetics 95
    David G. Nicholls and Marius W. Ward
    1 Introduction 95
    2 Mitochondrial membrane potential 95
    Monitoring dynamic changes in mitochondrial membrane potential with TMRM+ or
    rhodamine 123 97
    Interpretation of dynamic whole cell fluorescence traces obtained with TMRM+ or rhodamine 123 99
    Equilibrium monitoring ofmitochondrial membrane potential 99
    Cytoplasmic free Ca2+ concentration with fura-2 101
    Simultaneous monitoring with TMRM+ or rhodamine 123 of dynamic changes in k~m and cytoplasmic free calcium concentration 101
    3 Monitoringmitochondfialsuperoxidegeneration 103
    4 Monitofing hydrogen peroxide generation 104
    5 ATP/ADP ratios in cultured neurons 104
    References 105
    5 Targeting of nuclear-encoded proteins into and across the thylakoid membrane: isolation and analysis of intact chloroplasts and thylakoids from plants l07
    Colin Robinson and Alexandra Mant
    1 Introduction 107
    Choice of plants 108
    Growth conditions 108
    2 Isolation of intact chloroplasts 108
    3 In vitro synthesis of nuclear-encoded chloroplast proteins 113
    4 Import of proteins into isolated chloroplasts 114
    5 Import of proteins into isolated thylakoid membranes 118
    The import pathway for thylakoid lumen proteins 118
    The basic import assay 119
    Some variations on the basic assay 120
    Analysis of nuclear-encoded thylakoid membrane proteins 121
    References 125
    6 Transport into and out of the nucleus 127
    Patrizia Fanara, Adam C. Berger, Deanna M. Green, Henry Hagan, Michelle T. Harreman, Kavita A. Marfatia, B. Booth Quimby, Maureen A. Powers, and Anita H. Corbett
    1 Introduction 127
    2 Nuclear transport assays 128
    In vitro protein import in permeabilized cells 128
    In vivo NLS-GFP protein import assay 132
    Single nuclear pore transport assay 134
    In vivo transport studies using rnicroinjection into Xenopus oocytes 134
    In vivo RNA localization using fluorescence in situ hybridization (FISH) 136
    3 Nucleocytoplasmic protein shuttling assays 139
    Heterokaryon assays 139
    nup49 nuclear protein export assay in S. cerevisiae 141
    Antibody injection to detect protein shuttling 142
    4 Regulated nuclear transport: protein-protein interactions 143
    Ligand binding studies using overlay assays 143
    Fluorescence-based applications to map protein-protein interactions 147
    5 Genetic approaches to studying nuclear transport 148
    References 149
    7 Transport across the membrane of the endoplasmic reticulum l5l
    Karin R6misch
    1 Introduction 151
    2 Translocation into the ER and monitoring import 151
    3 Interaction with chaperones and protein folding 158
    4 Export of misfolded proteins from the ER to the cytosol for degradation 160
    References 162
    8 In vitro reconstitution of early to late endosome transport: biogenesis and subsequent fusion of transport intermediates 165
    Feng Gu and Jean Gruenberg
    1 Introduction 165
    2 Baby hamster kidney (BHK21) cell culture 167
    3 Labelling ofthe different endosomal compartments in BHK cells 168
    4 HomogenizationandsubcellularfractionationofBHK 170
    5 In vitro reconstitution of the ECV/MVB formation from early endosomes 172
    6 In vitro fusion of the ECV/MVBs with late endosomes 176
    7 Conclusion 179
    References 179
    9 Receptor biology 181
    Mercedes Dosil, Pamela Mentesana, and James B. Konopka
    1 Introduction 181
    2 Ligand binding assays 181
    General considerations 181
    Assaying ligand binding to membrane preparations 182
    Assaying ligand binding with whole cells 184
    Data analysis 185
    3 Epitope tags and fusion proteins 186
    Protein detection tags 186
    Chimeric receptors and GPCR-Gc~ fusion proteins 187
    4 Receptor modifications and receptor structure 188
    Post-translational modifications 188
    Receptor structure 189
    5 Receptor trafficking 190
    Visualization of receptor trafficking and analysis of receptor loss from the plasma membrane 191
    Membrane fractionation 191
    6 Heterologous expression ofreceptors in yeast 194
    Background 194
    General considerations 194
    Growth and transformation of yeast 195
    Analysis of receptor expression and activation 196
    References 197
    10 Measurement of signal transduction machinery 199
    Matthew Hodgkin, Michael Wakelam, and Carolyn Armour
    I Introduction 199
    2 Quantification ofphospholipase C (PLC) signalling in cells 199
    Measurement of PLC signal transduction pathway activity 200
    Protein kinase C assay 205
    Fluorescent quantification of changes in intracellular calcium concentration 207
    3 Quantification ofphospholipase D (PLD) activity in cells 208
    Measurement of DAG and ceramide mass 209
    4 Phosphatidylinositol 3-kinase (PI 3-kinase) signalling in cells 211
    Measurement ofPI 3-kinase downstream targets 212
    Quantification of3-phosphorylated lipids in cells 213
    5 Measurement of small G protein function in ceils 214
    Additional notes on the measurement of SMG protein activation 216
    6 Measurement of MAP kinase activation 217
    ERK kinase activity 217
    7 cAMP signalling in ceils 219
    8 Changes in cellular localization during signalling 220
    Changes in cytoskeletal architecture 222
    Visualization of signal transduction events in living cells 223
    References 223
    A1 List of suppliers 225
    Index 233