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A brief history of calcium imaging Andrew Hires Svoboda Lab Journal Club May 16th, 2008

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In the beginning, there were jellyfish <ul><li>SHIMOMURA O, JOHNSON FH, SAIGA Y. Extraction, purification and properties of aequorin , a bioluminescent protein from the luminous hydromedusan, Aequorea </li></ul><ul><li>J Cell Comp Physiol. 1962 Jun;59:223-39. </li></ul><ul><li>First calcium indicator was “genetically encoded” in vivo </li></ul><ul><li>Calcium sensitive, bioluminescent Aequorin was purified from Aequorea victoria </li></ul><ul><li>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.” </li></ul>

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Properties of Aequorin <ul><li>Cobinding of 3 Ca ++ sites gives blue fluorescence </li></ul><ul><li>Required cofactor, coelenterzine, destroyed from photon emission </li></ul><ul><li>Low light output. 12 Ca ++ ions used and 6 aqueorins destroyed per photon </li></ul><ul><li>Classically microinjected </li></ul><ul><li>Johnsn FH, Shimomura O. </li></ul><ul><li>Preparation and use of aequorin for rapid microdetermination of Ca 2+ inbiological systems. </li></ul><ul><li>Nat New Biol. 1972 Jun 28;237(78):287-8. </li></ul><ul><li>Now cloned. Coelenterzine is commercially available, membrane permeant </li></ul>

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Other techniques <ul><li>Ion selective electrodes </li></ul><ul><li>Rink TJ, Tsien RY, Warner AE. </li></ul><ul><li>Free calcium in Xenopus embryos measured with ion-selective microelectrodes. Nature. 1980 Feb 14;283(5748):658-60. </li></ul><ul><li>Metallochromic dyes </li></ul><ul><li>Fluorescent dyes </li></ul><ul><ul><li>BAPTA, quin2 - 1980 </li></ul></ul><ul><ul><li>fura2, indo - 1985 Tsien et. al </li></ul></ul><ul><ul><li>fluo, rhod - 1989 </li></ul></ul><ul><ul><li>Molecular Probes derivatives </li></ul></ul><ul><ul><ul><li>Kuhn 1993 </li></ul></ul></ul>

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Structures of some calcium dyes

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The first published almost GECI : FIP-CB SM <ul><li>BGFP-CM SM -RGFP </li></ul><ul><li>CaM not fused </li></ul><ul><li>Change induced by CaM-4Ca binding </li></ul><ul><li>Microinjected </li></ul>

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The first published GECI : Cameleon

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Early variants of cameleon

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Affinity engineering C1 YC2

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Subcellular targeting of Cameleons <ul><li>Intact YC2 : 74kD, cytosolic only </li></ul><ul><li>NLS-C2, NLS-YC3, nucleus only </li></ul><ul><li>YC3-ER, YC4-ER, ER only </li></ul><ul><li>Split CFP-CaM, M13-YFP (44, 30kD) </li></ul><ul><ul><li>Cytosol and nucleus </li></ul></ul>

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<ul><li>Replacement of EYFP with V68L/Q69K </li></ul><ul><li>YCX.1 series </li></ul><ul><li>Reduced pH sensitivity </li></ul><ul><li>Enhanced ratio change (Fmax/Fmin=2) </li></ul>

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Interference with split cameleons <ul><li>Pre-incubation of CaM with split YC2.1 abolished dynamic range </li></ul><ul><li>Linear fused YC less susceptible </li></ul>

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Intermolecular effects & buffering <ul><li>Little effect of high YC3.1 concentrations </li></ul><ul><li>Cells a,b = 150µM ; Cell c = 500µM blocks oscillations </li></ul>

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Insertion Schemes

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EYFP-Calmodulin insertion