A Brief History of Calcium Imaging
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Transcript of A Brief History of Calcium Imaging
A brief history of calcium imaging
Andrew HiresSvoboda Lab Journal Club
May 16th, 2008
In the beginning, there were jellyfish
SHIMOMURA O, JOHNSON FH, SAIGA Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea
J Cell Comp Physiol. 1962 Jun;59:223-39.
• First calcium indicator was “genetically encoded” in vivo• Calcium sensitive, bioluminescent Aequorin was purified from
Aequorea victoria
1st GFP ref in footnote! ”A protein giving solutions that look slightly greenish in sunlight through only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite, has also been isolated from squeezates.”
Properties of Aequorin
• Cobinding of 3 Ca++ sites gives blue fluorescence
• Required cofactor, coelenterzine, destroyed from photon emission
• Low light output. 12 Ca++ ions used and 6 aqueorins destroyed per photon
• Classically microinjectedJohnsn FH, Shimomura O.
Preparation and use of aequorin for rapid microdetermination of Ca 2+ inbiological systems.
Nat New Biol. 1972 Jun 28;237(78):287-8.
• Now cloned. Coelenterzine is commercially available, membrane permeant
Other techniques
• Ion selective electrodesRink TJ, Tsien RY, Warner AE.
Free calcium in Xenopus embryos measured with ion-selective microelectrodes. Nature. 1980 Feb 14;283(5748):658-60.
• Metallochromic dyes
• Fluorescent dyes– BAPTA, quin2 - 1980– fura2, indo - 1985 Tsien et. al– fluo, rhod - 1989– Molecular Probes derivatives
• Kuhn 1993
Structures of some calcium dyes
The first published almost GECI : FIP-CBSM
• BGFP-CMSM-RGFP
• CaM not fused
• Change induced by CaM-4Ca binding
• Microinjected
The first published GECI : Cameleon
Early variants of cameleon
Affinity engineering
C1
YC2
Subcellular targeting of Cameleons
• Intact YC2 : 74kD, cytosolic only• NLS-C2, NLS-YC3, nucleus only• YC3-ER, YC4-ER, ER only• Split CFP-CaM, M13-YFP (44, 30kD)
– Cytosol and nucleus
• Replacement of EYFP with V68L/Q69K• YCX.1 series• Reduced pH sensitivity• Enhanced ratio change (Fmax/Fmin=2)
Interference with split cameleons
• Pre-incubation of CaM with split YC2.1 abolished dynamic range
• Linear fused YC less susceptible
Intermolecular effects & buffering
• Little effect of high YC3.1 concentrations• Cells a,b = 150µM ; Cell c = 500µM blocks oscillations
Insertion Schemes
EYFP-Calmodulin insertion