A Brief History of Calcium Imaging

Slideshow Transcript

1. Slide 1: A brief history of calcium imaging Andrew Hires Svoboda Lab Journal Club May 16th, 2008
2. Slide 2: 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.”
3. Slide 3: 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 microinjected Johnsn 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
4. Slide 4: Other techniques • Ion selective electrodes Rink 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
5. Slide 5: Structures of some calcium dyes
6. Slide 6: The first published almost GECI : FIP-CBSM • BGFP-CMSM-RGFP • CaM not fused • Change induced by CaM-4Ca binding • Microinjected
7. Slide 7: The first published GECI : Cameleon
8. Slide 8: Early variants of cameleon
9. Slide 9: Affinity engineering C1 YC2
10. Slide 10: 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
11. Slide 11: • Replacement of EYFP with V68L/Q69K • YCX.1 series • Reduced pH sensitivity • Enhanced ratio change (Fmax/Fmin=2)
12. Slide 12: Interference with split cameleons • Pre-incubation of CaM with split YC2.1 abolished dynamic range • Linear fused YC less susceptible
13. Slide 13: Intermolecular effects & buffering • Little effect of high YC3.1 concentrations • Cells a,b = 150µM ; Cell c = 500µM blocks oscillations
14. Slide 15: Insertion Schemes
15. Slide 16: EYFP-Calmodulin insertion