简介
Summary:
Publisher Summary 1
Approaches and techniques are described for use in studying cell-cell interactions, focusing on biological processes where cell-cell interactions have been shown to play a critical role. Methodologies are given for working with specialized cell types, specialized domains of membrane contact, different developmental models of mammalian development, and distinct stages of medical importance. Protocols span molecular, cellular, biochemical, and physiological techniques. This second edition reflects recent developments in classical procedures. Fleming teaches in the School of Biological Sciences at the University of Southampton, UK. Annotation c. Book News, Inc., Portland, OR (booknews.com)
Publisher Summary 2
The book comprises ten chapters written by experts in the field on cell-cell interactions and their role in biology and medicine. Cell-cell interactions are the means by which cells are able to communicate, transfer information, develop spatial awareness and coordinate their differentiation. The ten areas have been selected for their breadth and relevance to modern research scientists where cell-cell interactions have been shown to play a critical role in biological processes. Chapters include methodologies for specialised cell types (keratinocytes, leukocytes, neurons, endothelial cells), specialised domains of membrane contact (adhesion molecules, gap junctions, tight junctions), different developmental models (Drosophila, Xenopus, mouse) and, for mammalian development, distinct stages of medical importance (oocyte-granulosa cells; preimplantation embryos; implantation). Throughout, molecular, cellular, biochemical and physiological protocols are provided in comprehensive detail to help the reader develop new skills and understanding.
目录
Preface p. v
Protocol list p. xiii
1 Cadherin adhesion regulation in keratinocytes Vania Braga p. 1
1 Introduction p. 1
2 Keratinocytes and cadherins p. 1
Background p. 1
Model p. 3
Advantages of the model p. 4
Disadvantages of the model p. 4
Keratinocyte cultures p. 5
3 Microinjection p. 7
Tools to modulate the activity of the small GTPases p. 7
Methodology p. 8
Strategy p. 8
Preparation of material to inject p. 10
Microinjection procedure to study keratinocyte junctions p. 11
Analysis of microinjection experiments p. 13
4 Immunostaining and confocal analysis p. 14
Detergent extraction p. 14
Immunolabelling p. 16
Confocal microscocopy p. 19
5 Cadherin functional assays p. 20
Inhibition by antibodies against E- and P-cadherin p. 20
Clustering cadherin receptors p. 21
6 Biochemical analysis of cadherin complexes p. 24
Cadherin and catenins p. 24
Protein extracts p. 26
Immunoprecipitation and Western blotting p. 27
7 Phosphorylation of cadherin complexes p. 30
Orthophosphate labelling p. 30
Isolation of phosphorylated proteins p. 31
Phosphopeptide mapping p. 33
Determination of the type of phosphorylated amino acid p. 34
2 Analysis of intercellular adhesion molecules in endothelial cells Gianfranco Bazzoni and Maria G. Lampugnani and Ofelia M. Martinez-Estrada and Elisabetta Dejana p. 37
1 Introduction p. 37
2 Endothelial cell culture p. 38
Human umbilical vein endothelial cells (HUVECs) p. 38
3 Vascular permeability and morphological changes associated with increased permeability p. 40
Vascular permeability p. 42
Immunofluorescence microscopy analysis of ECs p. 43
Acknowledgements p. 44
3 Gap junction-mediated interactions between cells David Becker and Colin Green p. 47
1 Introduction p. 47
2 Establishing the pattern and extent of communication between cells p. 47
Dye transfer p. 48
3 Gap junction density and connexin isoform expression p. 54
Immunohistochemistry: connexin-specific antibodies p. 54
Quantification of connexin plaque size and number p. 58
Expression analysis of mRNA by quantitative RT-PCR p. 59
Whole-mount in situ hybridization for connexins using digoxigenin p. 61
4 Manipulating gap junctional communication p. 63
Pharmacological agents p. 64
Antibody blockers p. 64
Extracellular loop peptides p. 65
Manipulation of gap junction gene expression p. 66
4 Functional analysis of the tight junction Marcellino Cereijido and Liora Shoshani and Ruben G. Contreras p. 71
1 Introduction p. 71
2 Model systems p. 72
Monolayers of epithelial cells p. 72
Co-cultures p. 74
3 Electron microscopy p. 77
Electron-dense tracers p. 78
Freeze-fracture p. 80
4 Information derived from the physical properties of permeating molecules p. 82
Solute size and charge assay for paracellular transport p. 82
5 The tight junction also functions as a fence p. 85
6 Analysis of second messenger regulation of tight junction sealing and activity p. 87
Ca[superscript 2+] signalling p. 87
Changes in phosphorylation of TJ-associated molecules p. 87
Acknowledgements p. 89
5 Leukocyte cell adhesion interactions Carl G. Gahmberg and Leena Valmu and Tiina J. Hilden and Eveliina Ihanus and Li Tian and Henrietta Nyman and Ulla Lehto and Asko Uppala and Tanja-Maria Ranta and Erkki Koivunen p. 93
1 Introduction p. 93
General p. 93
The leukocyte [beta subscript 2]-integrins p. 94
The ICAMs p. 94
The selectins p. 96
Other leukocyte adhesion molecules p. 96
2 Activation of leukocyte adhesion p. 97
Methods to activate integrins p. 97
3 Identification and preliminary characterization of leukocyte adhesion molecules p. 98
4 Phosphorylation of adhesion molecules p. 101
5 Isolation of active [beta subscript 2]-integrins p. 104
6 Preparation of I-domains p. 105
7 Construction and purification of Fc chimeric proteins p. 108
8 Cell adhesion assay p. 111
9 Detection of ICAMs p. 112
10 Use of phage display libraries for isolation of peptide ligands to [beta subscript 2]-integrins p. 113
Acknowledgements p. 117
6 Studying cell interactions during development of the nervous system in Drosophila David Shepherd and Louise Block and James Folwell and Darren Williams p. 119
1 Introduction p. 119
Why study Drosophila? p. 119
For the absolute beginner p. 120
2 Preparing tissues for study p. 120
Equipment required p. 121
Handling tissues p. 121
Dissections p. 121
3 Revealing neurons and glia p. 123
Reporter genes p. 123
Dye injections p. 126
Using antibodies to reveal specific neurons p. 130
Lineage tracing and birthdating of cells p. 135
4 Manipulation of cells p. 137
Methods of ectopic gene activation p. 137
The GAL4 enhancer-trap technique p. 139
Laser gene activation in single cells p. 140
Mosaic analysis p. 143
Methods of targeted cellular ablation p. 148
Acknowledgements p. 149
7 Tight junction protein expression in early Xenopus development and protein interaction studies Sandra Citi and Fabio D'Atri and Michelangelo Cordenonsi and Pietro Cardellini p. 153
1 Introduction p. 153
2 Tight junction formation during Xenopus laevis early development p. 153
Preparation of Xenopus laevis eggs and embryos p. 154
Whole-mount immunolocalization of tight junction proteins in Xenopus laevis eggs and early embryos p. 156
Scanning electron microscopic analysis of junctions in Xenopus embryos p. 159
Biochemical analysis of Xenopus eggs and embryos p. 160
Microinjection of Xenopus oocytes, eggs, and embryos p. 162
3 Assays to study protein-protein interactions in vitro p. 164
Bacterial and insect cell expression of recombinant proteins p. 165
Preparation of vertebrate cell lysates containing tight junction proteins p. 168
GST pull-down p. 169
Determination of the dissociation constant for protein-protein interaction (K[subscript d]) p. 171
Immunoprecipitation p. 173
Websites for specialized suppliers p. 175
Acknowledgements p. 175
8 Oocyte-granulosa cell interactions Barbara C. Vanderhyden p. 177
1 Introduction p. 177
2 Mammalian oocyte growth and development in vivo p. 177
3 Isolation and culture of pre-antral follicles and oocyte-granulosa cell complexes p. 179
Overview p. 179
Isolation and culture of rodent pre-antral follicles p. 180
Growth of pre-antral follicles from commercially important species p. 182
Growth of primate pre-antral follicles p. 183
Parameters indicative of successful follicle development p. 183
4 In vitro maturation techniques p. 183
Meiotic (nuclear) and cytoplasmic maturation of oocytes p. 183
Factors to consider when designing oocyte maturation experiments p. 184
In vitro maturation of rodent oocytes p. 185
In vitro maturation of oocytes from domestic species p. 186
In vitro maturation of primate oocytes p. 187
Evaluation of in vitro maturation p. 187
5 Evaluating granulosa cell effects on oocyte development p. 188
6 Evaluating oocyte effects on granulosa cell development p. 188
Isolation and culture of cumulus granulosa cells p. 189
Isolation and culture of mural granulosa cells p. 192
Granulosa cell differentiation in culture p. 192
7 Analysis of oocyte-granulosa cell gap junctional communication p. 193
8 Antisense and other strategies for interfering with oocyte-granulosa cell interactions p. 194
Manipulation of gene expression in oocytes p. 194
Manipulation of gene expression in granulosa cells p. 198
9 In vivo experimental systems for studying oocyte-granulosa interactions p. 198
Acknowledgements p. 198
9 Cell-cell interactions in early mammalian development Tom P. Fleming and Judith J. Eckert and Wing Yee Kwong and Fay C. Thomas and Daniel J. Miller and Irina Fesenko and Andrew Mears and Bhav Sheth p. 203
1 Introduction p. 203
Cell-cell interactions in blastocyst morphogenesis p. 203
2 Generation of synchronous embryos and cell clusters p. 206
3 Analyses of gene and protein expression p. 210
Gene expression analysis p. 210
Protein localization at cell contact sites in pre-implantation embryos p. 214
4 Analysis of cell lineage segregation p. 218
Acknowledgements p. 227
10 Model systems for the investigation of implantation John D. Aplin and Carlos Simon and Susan J. Kimber and Melanie J. Blissett and Helen Lacey and Chie-Pein Chen and Carolyn J. P. Jones p. 229
1 Embryo-endometrial interactions p. 229
Co-culture of human embryos with human endometrial epithelial cells as a model for implantation studies p. 229
Murine uterine epithelial cell cultures p. 235
2 Human placental development p. 243
Introduction: placental cell lineages p. 243
Cell culture methods p. 243
Extravillous trophoblast p. 244
Isolation and culture of placental fibroblasts p. 253
A1 List of suppliers p. 259
Index p. 265
Protocol list p. xiii
1 Cadherin adhesion regulation in keratinocytes Vania Braga p. 1
1 Introduction p. 1
2 Keratinocytes and cadherins p. 1
Background p. 1
Model p. 3
Advantages of the model p. 4
Disadvantages of the model p. 4
Keratinocyte cultures p. 5
3 Microinjection p. 7
Tools to modulate the activity of the small GTPases p. 7
Methodology p. 8
Strategy p. 8
Preparation of material to inject p. 10
Microinjection procedure to study keratinocyte junctions p. 11
Analysis of microinjection experiments p. 13
4 Immunostaining and confocal analysis p. 14
Detergent extraction p. 14
Immunolabelling p. 16
Confocal microscocopy p. 19
5 Cadherin functional assays p. 20
Inhibition by antibodies against E- and P-cadherin p. 20
Clustering cadherin receptors p. 21
6 Biochemical analysis of cadherin complexes p. 24
Cadherin and catenins p. 24
Protein extracts p. 26
Immunoprecipitation and Western blotting p. 27
7 Phosphorylation of cadherin complexes p. 30
Orthophosphate labelling p. 30
Isolation of phosphorylated proteins p. 31
Phosphopeptide mapping p. 33
Determination of the type of phosphorylated amino acid p. 34
2 Analysis of intercellular adhesion molecules in endothelial cells Gianfranco Bazzoni and Maria G. Lampugnani and Ofelia M. Martinez-Estrada and Elisabetta Dejana p. 37
1 Introduction p. 37
2 Endothelial cell culture p. 38
Human umbilical vein endothelial cells (HUVECs) p. 38
3 Vascular permeability and morphological changes associated with increased permeability p. 40
Vascular permeability p. 42
Immunofluorescence microscopy analysis of ECs p. 43
Acknowledgements p. 44
3 Gap junction-mediated interactions between cells David Becker and Colin Green p. 47
1 Introduction p. 47
2 Establishing the pattern and extent of communication between cells p. 47
Dye transfer p. 48
3 Gap junction density and connexin isoform expression p. 54
Immunohistochemistry: connexin-specific antibodies p. 54
Quantification of connexin plaque size and number p. 58
Expression analysis of mRNA by quantitative RT-PCR p. 59
Whole-mount in situ hybridization for connexins using digoxigenin p. 61
4 Manipulating gap junctional communication p. 63
Pharmacological agents p. 64
Antibody blockers p. 64
Extracellular loop peptides p. 65
Manipulation of gap junction gene expression p. 66
4 Functional analysis of the tight junction Marcellino Cereijido and Liora Shoshani and Ruben G. Contreras p. 71
1 Introduction p. 71
2 Model systems p. 72
Monolayers of epithelial cells p. 72
Co-cultures p. 74
3 Electron microscopy p. 77
Electron-dense tracers p. 78
Freeze-fracture p. 80
4 Information derived from the physical properties of permeating molecules p. 82
Solute size and charge assay for paracellular transport p. 82
5 The tight junction also functions as a fence p. 85
6 Analysis of second messenger regulation of tight junction sealing and activity p. 87
Ca[superscript 2+] signalling p. 87
Changes in phosphorylation of TJ-associated molecules p. 87
Acknowledgements p. 89
5 Leukocyte cell adhesion interactions Carl G. Gahmberg and Leena Valmu and Tiina J. Hilden and Eveliina Ihanus and Li Tian and Henrietta Nyman and Ulla Lehto and Asko Uppala and Tanja-Maria Ranta and Erkki Koivunen p. 93
1 Introduction p. 93
General p. 93
The leukocyte [beta subscript 2]-integrins p. 94
The ICAMs p. 94
The selectins p. 96
Other leukocyte adhesion molecules p. 96
2 Activation of leukocyte adhesion p. 97
Methods to activate integrins p. 97
3 Identification and preliminary characterization of leukocyte adhesion molecules p. 98
4 Phosphorylation of adhesion molecules p. 101
5 Isolation of active [beta subscript 2]-integrins p. 104
6 Preparation of I-domains p. 105
7 Construction and purification of Fc chimeric proteins p. 108
8 Cell adhesion assay p. 111
9 Detection of ICAMs p. 112
10 Use of phage display libraries for isolation of peptide ligands to [beta subscript 2]-integrins p. 113
Acknowledgements p. 117
6 Studying cell interactions during development of the nervous system in Drosophila David Shepherd and Louise Block and James Folwell and Darren Williams p. 119
1 Introduction p. 119
Why study Drosophila? p. 119
For the absolute beginner p. 120
2 Preparing tissues for study p. 120
Equipment required p. 121
Handling tissues p. 121
Dissections p. 121
3 Revealing neurons and glia p. 123
Reporter genes p. 123
Dye injections p. 126
Using antibodies to reveal specific neurons p. 130
Lineage tracing and birthdating of cells p. 135
4 Manipulation of cells p. 137
Methods of ectopic gene activation p. 137
The GAL4 enhancer-trap technique p. 139
Laser gene activation in single cells p. 140
Mosaic analysis p. 143
Methods of targeted cellular ablation p. 148
Acknowledgements p. 149
7 Tight junction protein expression in early Xenopus development and protein interaction studies Sandra Citi and Fabio D'Atri and Michelangelo Cordenonsi and Pietro Cardellini p. 153
1 Introduction p. 153
2 Tight junction formation during Xenopus laevis early development p. 153
Preparation of Xenopus laevis eggs and embryos p. 154
Whole-mount immunolocalization of tight junction proteins in Xenopus laevis eggs and early embryos p. 156
Scanning electron microscopic analysis of junctions in Xenopus embryos p. 159
Biochemical analysis of Xenopus eggs and embryos p. 160
Microinjection of Xenopus oocytes, eggs, and embryos p. 162
3 Assays to study protein-protein interactions in vitro p. 164
Bacterial and insect cell expression of recombinant proteins p. 165
Preparation of vertebrate cell lysates containing tight junction proteins p. 168
GST pull-down p. 169
Determination of the dissociation constant for protein-protein interaction (K[subscript d]) p. 171
Immunoprecipitation p. 173
Websites for specialized suppliers p. 175
Acknowledgements p. 175
8 Oocyte-granulosa cell interactions Barbara C. Vanderhyden p. 177
1 Introduction p. 177
2 Mammalian oocyte growth and development in vivo p. 177
3 Isolation and culture of pre-antral follicles and oocyte-granulosa cell complexes p. 179
Overview p. 179
Isolation and culture of rodent pre-antral follicles p. 180
Growth of pre-antral follicles from commercially important species p. 182
Growth of primate pre-antral follicles p. 183
Parameters indicative of successful follicle development p. 183
4 In vitro maturation techniques p. 183
Meiotic (nuclear) and cytoplasmic maturation of oocytes p. 183
Factors to consider when designing oocyte maturation experiments p. 184
In vitro maturation of rodent oocytes p. 185
In vitro maturation of oocytes from domestic species p. 186
In vitro maturation of primate oocytes p. 187
Evaluation of in vitro maturation p. 187
5 Evaluating granulosa cell effects on oocyte development p. 188
6 Evaluating oocyte effects on granulosa cell development p. 188
Isolation and culture of cumulus granulosa cells p. 189
Isolation and culture of mural granulosa cells p. 192
Granulosa cell differentiation in culture p. 192
7 Analysis of oocyte-granulosa cell gap junctional communication p. 193
8 Antisense and other strategies for interfering with oocyte-granulosa cell interactions p. 194
Manipulation of gene expression in oocytes p. 194
Manipulation of gene expression in granulosa cells p. 198
9 In vivo experimental systems for studying oocyte-granulosa interactions p. 198
Acknowledgements p. 198
9 Cell-cell interactions in early mammalian development Tom P. Fleming and Judith J. Eckert and Wing Yee Kwong and Fay C. Thomas and Daniel J. Miller and Irina Fesenko and Andrew Mears and Bhav Sheth p. 203
1 Introduction p. 203
Cell-cell interactions in blastocyst morphogenesis p. 203
2 Generation of synchronous embryos and cell clusters p. 206
3 Analyses of gene and protein expression p. 210
Gene expression analysis p. 210
Protein localization at cell contact sites in pre-implantation embryos p. 214
4 Analysis of cell lineage segregation p. 218
Acknowledgements p. 227
10 Model systems for the investigation of implantation John D. Aplin and Carlos Simon and Susan J. Kimber and Melanie J. Blissett and Helen Lacey and Chie-Pein Chen and Carolyn J. P. Jones p. 229
1 Embryo-endometrial interactions p. 229
Co-culture of human embryos with human endometrial epithelial cells as a model for implantation studies p. 229
Murine uterine epithelial cell cultures p. 235
2 Human placental development p. 243
Introduction: placental cell lineages p. 243
Cell culture methods p. 243
Extravillous trophoblast p. 244
Isolation and culture of placental fibroblasts p. 253
A1 List of suppliers p. 259
Index p. 265
Cell-cell interactions. 2nd ed. /
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