Essential Cell Biology A Practical Approach Volume 2: Cell Function

by Davey, John; Lord, J. Michael

• ISBN13: 9780199638321
• ISBN10: 0199638322
• Format: Hardcover
• Copyright: 2003-08-28
• Publisher: Oxford University Press

Summary

Biological and medical research relies upon an integrated understanding of the molecules within cells and of the interactions between cells. This has imposed great demands on investigators. Being an expert in a relatively narrow area is no longer sufficient as many studies now require theuse of a wide range of techniques to provide the necessary integration. A lack of familiarity with the experimental possibilities can make such diversification difficult to achieve. This two volume set of Essential Cell Biology is designed to help researchers overcome these problems. It has notbeen possible to include all of the techniques available in cell biology so the challenge was to identify those that might be most relevant to researchers who are new to this topic. We have tried to cover both traditional and more recent approaches. The theory and basic principles of each techniqueare described, together with detailed protocols and advice for trouble shooting. Directions to more specialised techniques are also included. We hope the result inspires readers to experience the challenges and rewards of cell biology research for themselves and to contribute to the ongoing taskof understanding the life of the cell.Essential Cell Biology volume 2 concentrates on understanding how the cell functions whilst Volume 1 focuses on techniques for studying cell structure. Volume 2 details the essential background information and protocols for investigating cell cell function, including, for example, investigations ofcellular transport, receptors and gene expression. This is the essential guide to cell biology for researchers new to the field.

Table of Contents

List of protocols	xi
Abbreviations	xv
1 Enzyme activities	1	(46)
Keith F. Tipton
1 Introduction	1	(1)
2 Enzyme specificity and nomenclature	1	(1)
3 Determination of enzyme activity	1	(4)
Reaction progress curves	2	(1)
The effects of enzyme concentration and expression of activities	3	(1)
Conditions for activity measurements	4	(1)
4 The effects of substrate concentration	5	(4)
The Michaelis-Menten relationship	5	(1)
Failure to obey the Michaelis-Menten equation	5	(4)
5 Enzyme inhibitors	9	(31)
Reversible inhibitors	9	(17)
Tight-binding inhibitors	26	(3)
Irreversible inhibitors	29	(11)
6 Behaviour in vivo	40	(4)
Recovery from irreversible inhibition	40	(1)
Effects of cellular processes	41	(2)
Inhibitor specificity	43	(1)
References	44	(3)
2 Gene expression	47	(32)
Luis Parada, Cem Elbi, Miroslav Dundr, and Tom Mistelf
1 Introduction	47	(1)
2 Indirect immunofluorescence	48	(3)
3 GFP in fixed or living cells	51	(2)
4 Nuclear matrix preparation	53	(2)
5 Visualization of transcription sites	55	(4)
6 Fluorescence in situ hybridization	59	(8)
7 Chromosome preparation and staining	67	(3)
8 Fluorescent chromosome painting	70	(6)
References	76	(3)
3 Regulation of the cell cycle	79	(16)
Diana M. Gitig and Andrew Koff
1 Introduction	79	(1)
2 Cell biology: detection of cell cycle arrests	79	(4)
3 Genetics: knockout and transgenic mice	83	(2)
4 Biochemistry	85	(6)
In vitro: recombinant proteins	85	(1)
In vivo: extract reconstitution assays	86	(5)
5 Conclusions	91	(1)
Acknowledgements	91	(1)
References	91	(4)
4 Cellular bioenergetics	95	(12)
David G. Nicholls and Manus W. Ward
1 Introduction	95	(1)
2 Mitochondrial membrane potential	95	(8)
Monitoring dynamic changes in mitochondrial membrane potential with TMRM+ or rhodamine 123	97	(2)
Interpretation of dynamic whole cell fluorescence traces obtained with TMRM+ or rhodamine 123	99	(1)
Equilibrium monitoring of mitochondrial membrane potential	99	(2)
Cytoplasmic free Cat+ concentration with fura-2	101	(1)
Simultaneous monitoring with TMRM+ or rhodamine 123 of dynamic changes in m and cytoplasmic free calcium concentration	101	(2)
3 Monitoring mitochondrial superoxide generation	103	(1)
4 Monitoring hydrogen peroxide generation	104	(1)
5 ATP/ADP ratios in cultured neurons	104	(1)
References	105	(2)
5 Targeting of nuclear-encoded proteins into and across the thylakoid membrane: isolation and analysis of intact chloroplasts and thylakoids from plants	107	(20)
Colin Robinson and Alexandra Mant
1 Introduction	107	(1)
Choice of plants 108 Growth conditions	108	(1)
2 Isolation of intact chloroplasts	108	(5)
3 In vitro synthesis of nuclear-encoded chloroplast proteins	113	(1)
4 Import of proteins into isolated chloroplasts	114	(4)
5 Import of proteins into isolated thylakoid membranes	118	(7)
The import pathway for thylakoid lumen proteins	118	(1)
The basic import assay	119	(1)
Some variations on the basic assay	120	(1)
Analysis of nuclear-encoded thylakoid membrane proteins	121	(4)
References	125	(2)
6 Transport into and out of the nucleus	127	(24)
Patrizia Fanara, Adam C. Berger, Deanna M. Green, Henry Hagan, Michelle T. Harpeman, Kovita A. Morfatia, B. Booth Quimby, Maureen A. Powers, and Anita H. Corbett
1 Introduction	127	(1)
2 Nuclear transport assays	128	(11)
In vitro protein import in permeabilized cells	128	(4)
In vivo NLS-GFP protein import assay	132	(2)
Single nuclear pore transport assay	134	(1)
In vivo transport studies using microinjection into Xenopus oocytes	134	(2)
In vivo RNA localization using fluorescence in situ hybridization (FISH)	136	(3)
3 Nucleocytoplasmic protein shuttling assays	139	(4)
Heterokaryon assays	139	(2)
nup49 nuclear protein export assay in S. cerevisiae	141	(1)
Antibody injection to detect protein shuttling	142	(1)
4 Regulated nuclear transport: protein-protein interactions	143	(5)
Ligand binding studies using overlay assays	143	(4)
Fluorescence-based applications to map protein-protein interactions	147	(1)
5 Genetic approaches to studying nuclear transport	148	(1)
References	149	(2)
7 Transport across the membrane of the endoplasmic reticulum	151	(14)
Karin Römisch
1 Introduction	151	(1)
2 Translocation into the ER and monitoring import	151	(7)
3 Interaction with chaperones and protein folding	158	(2)
4 Export of misfolded proteins from the ER to the cytosol for degradation	160	(2)
References	162	(3)
8 In vitro reconstitution of early to late endosome transport: biogenesis and subsequent fusion of transport intermediates	165	(16)
Feng Gu and Jean Greenberg
1 Introduction	165	(2)
2 Baby hamster kidney (BHK21) cell culture	167	(1)
3 Labelling of the different endosomal compartments in BHK cells	168	(2)
4 Homogenization and subcellular fractionation of BHK	170	(2)
5 In vitro reconstitution of the ECV/MVB formation from early endosomes	172	(4)
6 In vitro fusion of the ECV/MVBs with late endosomes	176	(3)
7 Conclusion	179	(1)
References	179	(2)
9 Receptor biology	181	(18)
Mercedes Dosil, Pamela Mentesana, and James B. Konopko
1 Introduction	181	(1)
2 Ligand binding assays	181	(5)
General considerations	181	(1)
Assaying ligand binding to membrane preparations	182	(2)
Assaying ligand binding with whole cells	184	(1)
Data analysis	185	(1)
3 Epitope tags and fusion proteins	186	(2)
Protein detection tags	186	(1)
Chimeric receptors and GPCR-Ga fusion proteins	187	(1)
4 Receptor modifications and receptor structure	188	(2)
Post-translational modifications	188	(1)
Receptor structure	189	(1)
5 Receptor trafficking	190	(4)
Visualization of receptor trafficking and analysis of receptor loss from the plasma membrane	191	(1)
Membrane fractionation	191	(3)
6 Heterologous expression of receptors in yeast	194	(3)
Background	194	(1)
General considerations	194	(1)
Growth and transformation of yeast	195	(1)
Analysis of receptor expression and activation	196	(1)
References	197	(2)
10 Measurement of signal transduction machinery	199	(26)
Matthew Hodgkin, Michael Wakelam, and Carolyn Armour
1 Introduction	199	(1)
2 Quantification of phospholipase C (PLC) signalling in cells	199	(9)
Measurement of PLC signal transduction pathway activity	200	(5)
Protein kinase C assay	205	(2)
Fluorescent quantification of changes in intracellular calcium concentration	207	(1)
3 Quantification of phospholipase D (PLD) activity in cells	208	(3)
Measurement of DAG and ceramide mass	209	(2)
4 Phosphatidylinositol 3-kinase (PI 3-kinase) signalling in cells	211	(3)
Measurement of PI 3-kinase downstream targets	212	(1)
Quantification of 3-phosphorylated lipids in cells	213	(1)
5 Measurement of small G protein function in cells	214	(3)
Additional notes on the measurement of SMG protein activation	216	(1)
6 Measurement of M P kinase activation	217	(2)
ERK kinase activity	217	(2)
7 cAMP signalling in cells	219	(1)
8 Changes in cellular localization during signalling	220	(3)
Changes in cytoskeletal architecture	222	(1)
Visualization of signal transduction events in living cells	223	(1)
References	223	(2)
A1 List of suppliers	225	(8)
Index	233