Calcium Signalling A Practical Approach

by Tepikin, Alexei

• ISBN13: 9780199638482
• ISBN10: 0199638489
• Edition: 2nd
• Format: Hardcover
• Copyright: 2001-03-01
• Publisher: Oxford University Press

Summary

This new edition of Calcium Signalling focuses on the applications of fluorescent and bioluminescent techniques for studying calcium signalling and calcium dependent processes. Cameleon probes, targeting bioluminescent probes, low affinity fluorescence indicators and chelators, and photometrictechniques are all covered in depth. Also described in detail are methods for monitoring mitochondrial function, measuring calcium in the nuclear envelope and nucleoplasm, measuring calcium dependent gene expression, monitoring calcium signals in multicellular preparations, and measuring calciumextrusion. A major problem of fluorescent calcium measurements is the artifacts from zinc and transition metals and a chapter is devoted to their detection and minimization. Some of the pharmacological methods of studying calcium release are also covered. Many of the chapters are written by thescientists who developed the methods they are describing so the authors could not be more qualified. It is hoped that this volume will prove as invaluable to the studying cellular calcium as its predecessor.

Table of Contents

Preface	v
List of protocols	xv
Abbreviations	xix
Part One New probes, new instruments, new methods
Cameleons as cytosolic and intra-organellar calcium probes	3	(14)
Atsushi Miyawaki
Juan Llopis
Hideaki Mizuno
Kees Jalink
Roger Y. Tsien
Introduction	3	(1)
Cameleons as new generation indicators for Ca2+	3	(2)
Advantages of cameleons over synthetic fluorescent calcium chelators and photoprotein aequorin	3	(1)
Evolutions of cameleons	4	(1)
Measurements of [Ca2+] (cytosolic Ca2+ concentration) using improved yellow camelons	5	(8)
Improved yellow cameleons (yellow cameleons-2.1 and -3.1)	5	(3)
Concentration of yellow cameleons inside cells	8	(3)
Constitutive expression of cameleons and interaction with CaM and CaM-dependent enzymes	11	(1)
Photochromism of yellow fluorescent protein	11	(2)
Measurements of nuclear Ca2+ concentration ([Ca2+]n)	13	(1)
[Ca2+]c versus [Ca2+]n	13	(1)
Targeting yellow cameleon-3.1 to nucleus	13	(1)
Sectional images of [Ca2+]c and [Ca2+]n by two-photon excitation	14	(1)
Measurements of free Ca2+ concentration inside the endoplasmic reticulum ([Ca2+]er)	14	(3)
References	15	(2)
Photometry, video imaging, confocal and multi-photon microscopy approaches in calcium signalling studies	17	(28)
Peter Lipp
Martin D. Bootman
Tony Collins
General introduction	17	(1)
Fluorescent Ca2+ indicators	17	(5)
Loading Ca2+ indicators	18	(2)
Choice of Ca2+ indicator	20	(2)
Using adherent cells	22	(1)
Photometry and video imaging of Ca2+	22	(4)
Photomultiplier tubes	22	(1)
Cameras	23	(1)
Data format--counts and images	24	(1)
Intensity depth	24	(1)
Look-up tables	24	(1)
Photometry versus video imaging	25	(1)
Brightness and gain-settings	25	(1)
Troubleshooting photometry and video imaging	26	(2)
Image too dark	26	(1)
Image too bright	27	(1)
Indicator bleaching	27	(1)
Single-photon laser scanning confocal microscopy	28	(1)
General introduction	28	(1)
General arrangement of a laser scanning confocal microscope	28	(1)
Spinning disc confocal microscopes	29	(1)
Advantages and pitfalls of confocal microscopy	29	(4)
Speed considerations	29	(2)
Problems with changing cell shape	31	(1)
Intensity depth, gain, and off-set	31	(2)
Examples	33	(5)
Linear Ca2+ waves in image mode	33	(2)
Linear Ca2+ wave in line scan mode	35	(1)
Elementary Ca2+ signals	36	(2)
Multi-photon confocal microscopy	38	(2)
Background	38	(1)
Hardware	39	(1)
Multi-photon photolysis	40	(1)
Data processing	41	(4)
Calibration of the indicator fluorescence	41	(2)
Self-ratio of single-wavelength indicators	43	(1)
Pseudocolour	44	(1)
Archiving	44	(1)
References	44	(1)
Detecting and minimizing errors in calcium-probe measurements arising from transition metals and zinc	45	(14)
Vladislav Snitsarev
Alan R. Kay
Introduction	45	(2)
Competition between endogenous transition metals and Ca2+ for fluorescent probes	47	(1)
Membrane-permeant transition metal chelators	48	(1)
Determining if fluorometric measurements of Ca2+ have been perturbed by transition metal ions	48	(11)
Acknowledgements	55	(1)
References	55	(4)
Targeting of bioluminescent probes and calcium measurements in different subcellular compartments	59	(20)
Paulo J. Magalhaes
Marisa Brini
Paolo Pinton
Luisa Filippin
Anna Chiesa
Tullio Pozzan
Rosario Rizzuto
Introduction	59	(1)
Background principles	60	(1)
Designing chimeric aequorin variants	61	(9)
Bioluminescent probes for mitochondrial matrix and mitochondrial intermembrane space	66	(1)
Measurement of ER and SR calcium using targeted aequorin	67	(1)
Targeted aequorin as an instrument of calcium measurement in the Golgi apparatus	68	(1)
Cytoplasmic and plasma membrane targeted bioluminescent probes	69	(1)
Nuclear targeting of aequorin	69	(1)
Use of aequorin in the presence of high Ca2+ concentrations	70	(2)
Concluding practical considerations	72	(7)
References	74	(5)
Part Two Calcium measurement in different organelles
Monitoring mitochondrial function in single cells	79	(28)
Mart H. Mojet
D. Jake Jacobson
Julie Keelan
Olga Vergun
Michael R. Duchen
Introduction	79	(1)
Historical perspective: why is mitochondrial function interesting in the context of a book on calcium signalling?	79	(1)
Fundamentals	80	(3)
Mitochondrial metabolism	80	(1)
The relationship between ΔY and redox state	81	(1)
Consequences of mitochondrial depolarization: mitochondria as ATP consumers	82	(1)
Fluorescence measurement and imaging of mitochondrial function	83	(1)
NADH and flavoprotein autofluorescence	83	(3)
Theoretical basis	83	(1)
Protocols	84	(2)
Validation and limitations	86	(1)
Fluorimetric measurement of mitochondrial potential	86	(8)
Theoretical basis	86	(2)
Approaches to the use of indicators and technical requirements	88	(1)
Calibration	89	(1)
Validation	90	(3)
Toxicity	93	(1)
An interesting question?	93	(1)
Measuring mitochondrial calcium ([Ca2+]mt)	94	(5)
Introduction and theoretical basis	94	(1)
Protocols	95	(3)
Validation and limitations	98	(1)
Measuring changes in [ATP]	99	(2)
Luciferase	100	(1)
[Mg2+]i	100	(1)
Cytochrome absorptance measurements	101	(2)
Theoretical basis	101	(1)
Methods and limitations	101	(1)
Perspectives	102	(1)
Simultaneous measurements of mitochondrial and other cellular parameters	103	(4)
Simultaneous monitoring of Δψm and [Ca2+]I	103	(1)
Simultaneous recording of Δψm and autofluorescence	104	(1)
Simultaneous recording of [Ca2+]cyt and [Ca2+]mt	104	(1)
References	105	(2)
Appendix	107	(108)
The mitochondriac's basic pharmacopoeia	107	(1)
Inhibitors of mitochondrial respiration	107	(4)
Complex I	107	(1)
Complex III	107	(1)
Complex IV	107	(1)
Uncouplers	108	(1)
The F1F0-ATP(synth)ase	108	(1)
Electron donors	108	(1)
The adenine nucleotide translocase	109	(1)
The permeability transition pore	109	(1)
The calcium uniporter	110	(1)
The Na+/Ca2+ exchanger	110	(1)
Using low-affinity fluorescent calcium indicators and chelators for monitoring and manipulating free [Ca2+] in the endoplasmic reticulum	111	(14)
Aldebaran M. Hofer
Introduction	111	(3)
Basic principles	112	(1)
Mechanism of dye uptake	112	(1)
Choice of indicator	113	(1)
Experimental protocols (permeabilized cells)	114	(4)
Notes on calcium buffers	115	(1)
Experiments in patch clamped cells	116	(1)
Intact cell measurements	116	(2)
Calibration procedures	118	(1)
Pitfalls	118	(1)
Problems of multiple compartments	118	(1)
Interference by Mg2+ and other ions	119	(1)
Advantages	119	(1)
Manipulating intrastore [Ca2+] with a membrane-permeant Ca2+ chelator, TPEN	119	(6)
Practical considerations for using TPEN	122	(1)
Applications	122	(1)
Acknowledgements	123	(1)
References	123	(2)
Measuring calcium in the nuclear envelope and nucleoplasm	125	(14)
Oleg Gerasimenko
Julia Gerasimenko
Introduction	125	(1)
Techniques and experiments described in the chapter	125	(1)
Calcium transport in the nucleus	125	(1)
Experimenting with isolated nuclei	126	(6)
Preparation of isolated mouse liver nuclei	126	(1)
Fluorescent labelling of nucleoplasm and nuclear envelope of isolated nuclei: measuring calcium signals in the nuclear envelope and nucleoplasm	127	(5)
Measurements of nuclear and cytosolic Ca2+ signals in intact isolated cells	132	(7)
References	135	(4)
Part Three Monitoring specific calcium reactions
Controlling cytoplasmic calcium and measuring calcium-dependent gene expression in intact cells	139	(16)
Ricardo E. Dolmetsch
Paul A. Negulescu
Introduction	139	(1)
Measuring gene expression in single cells	140	(5)
Reporter genes	140	(5)
Generating defined cytoplasmic [Ca2+]i signals	145	(10)
Constructing a calcium clamp	145	(4)
Using calcium clamp	149	(4)
Characterizing and troubleshooting the Ca2+ clamp	153	(1)
Summary and future perspectives	154	(1)
References	154	(1)
Monitoring the generation and propagation of calcium signals in multicellular preparations	155	(22)
Lowrence D. Gaspers
Sandip Patel
Anthony J. Morgan
Paul A. Anderson
Andrew P. Thomas
Introduction	155	(2)
Perfusion apparatus and imaging systems	157	(7)
Perfusion system	157	(1)
Membrane lung	158	(2)
Mixing chamber/bubble trap	160	(1)
Imaging chamber	161	(1)
Imaging devices	162	(2)
Perfusion	164	(2)
Perfusion media	164	(1)
Tissue isolation	165	(1)
Loading Ca2+ indicator dyes	166	(2)
Loading protocol	166	(1)
Dye retention	167	(1)
Photobleaching	168	(1)
Calibration	168	(2)
Calibration protocol	168	(2)
Image acquisition and analysis	170	(3)
Image quality	170	(1)
Deconvolving non-confocal fura-2 images	170	(3)
Future directions	173	(4)
Acknowledgements	174	(1)
References	174	(3)
Pharmacological studies of new calcium release mechanisms	177	(20)
Antony Galione
Justyn Thomas
Grant Churchill
Introduction	177	(1)
Preparation of sea urchin egg homogenates and microsomes	177	(2)
Collection of sea urchin eggs	177	(1)
Sea urchin egg homogenates	178	(1)
Fractionation of sea urchin egg homogenate by Percoll density gradient	179	(1)
Studying activation of calcium release channels	179	(7)
Fluorimetry of Ca2+ release from egg homogenates	179	(2)
Testing microsomal fractions for Ca2+ releasing properties	181	(1)
Pharmacology of cADPR signalling	182	(2)
Studying NAADP-induced Ca2+ release	184	(1)
Desensitization of NAADP-induced calcium release	184	(2)
Measuring production and degradation of second messengers that trigger calcium signals	186	(7)
Enzymatic synthesis and degradation of cADPR	186	(1)
NAADP metabolism	186	(3)
Detection of cADPR and NAADP metabolism by discontinuous bioassay	189	(1)
NGD assay: continuous assay for GDP-ribosyl cyclase activity	189	(2)
HPLC	191	(1)
Measurement of cADPR levels in cells and tissues	191	(2)
Binding of agonists to calcium release channels	193	(4)
cADPR binding studies	193	(1)
The NAADP receptor	194	(1)
References	195	(2)
Measuring calcium extrusion	197	(18)
Alexei V. Tepikin
Introduction	197	(1)
The droplet technique	198	(11)
Instruments and materials for the droplet technique	198	(1)
Droplet formation	199	(2)
Injection of substances into the droplet	201	(1)
Measurements of fluorescence changes of intracellular and extracellular indicators and calibration of calcium concentrations in the droplet experiment	202	(4)
Measuring droplet volume and volume of cells' calculation of calcium flux, rate of intracellular calcium concentration changes, and calcium binding capacity of the cytoplasm of the cell	206	(3)
Calcium-sensitive jam technique---using calcium indicators bound to dextrans to measure calcium extrusion	209	(6)
Instruments and materials for calcium-sensitive jam technique	209	(3)
Calcium-sensitive gel	212	(1)
References	212	(3)
A1 Fluorescent calcium indicators and caged calcium probes discussed in this volume	215	(4)
A2 List of suppliers	219	(6)
Index	225