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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
