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ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang

ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang. Artemis uncages the messenger. Physiol. Rev. (1987) 67:583. Light sensitive chymotrypsin inhibitor: an early ‘caged’ compound. Kaufman, Vratsanos & Erlanger (1968) Science 162:1487. Design principles of caged compounds.

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ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang

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  1. ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDSSamuel S.-H. Wang

  2. Artemis uncages the messenger Physiol. Rev. (1987) 67:583

  3. Light sensitive chymotrypsin inhibitor:an early ‘caged’ compound Kaufman, Vratsanos & Erlanger (1968) Science 162:1487

  4. Design principles of caged compounds H. Lester and J. Nerbonne (1982) Ann. Rev. Biophys. Bioeng. 11:151

  5. Adams and Tsien (1993)

  6. The dark reaction Decay of the aci-nitro intermediate of NPE-caged ATP J.W. Walker et al.(1988) JACS 110:7170

  7. Fast temporal control:caged calcium at the squid giant synapse K.R. Delaney and R.S. Zucker (1990) J.Physiol. 426:473

  8. Temporal dissection of signal kinetics Delays in Ca release after IP3 uncaging K. Khodakhah and D. Ogden (1993) PNAS 90:4976 Note: 1) [IP3]-dependent delay in Ca rise and IK(Ca); 2) phosphorescence artifact

  9. In practice, most caged compounds marketed have pretty fast dark reaction. A more variable quantity is the effectiveness with which caged compounds use light. The uncagability index depends on: Absorption (Tends to be constant for a given cage group) Quantum yield (Varies with modified molecule) Judging a caged compound

  10. Absorption spectra of some caged nucleotides J. Nerbonne (1986) Design and application of photolabile intracellular probes. In: Optical methods in cell physiology, ed. P. De Weer and B.M. Salzberg.

  11. Focal uncaging Wang and Augustine (1995)

  12. Two-photon excitation: the third dimension of resolution

  13. Caged fluorescein dextran

  14. Uncaging in single dendritic spines Svoboda, Tank & Denk (1996) Science 272:716

  15. Wang and Augustine (1995)

  16. Furuta et al. (1999) PNAS 96:1193

  17. Comparison of a new caging group,6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc), with previous caged compounds

  18. Scanning two-photon uncaging of glutamate

  19. Achieving a multiphoton effect by chemical means A new design principle: multiple-site caging Reduction of effective spontaneous hydrolysis Effective cross-section is MUCH larger (109-fold) than true two-photon excitation Chemical two-photon uncaging

  20. Chemical two-photon uncaging

  21. Improved axial resolution viachemical two-photon uncaging

  22. Wang, Khiroug and Augustine (2000) PNAS 97:8635

  23. Wang, Khiroug and Augustine (2000) PNAS 97:8635

  24. LTD induction causes a spreading decrease in receptor sensitivity

  25. Double-caged IP3 Goal: uncaging IP3 in single dendritic spines. S.E. Gelber, J.W. Walker, S.S.-H. Wang

  26. Wang, Denk and Hausser (2000), Nature Neuroscience 3:1266

  27. Block of calcium release differentiates sparse-PF and dense-PF coincidence mechanisms

  28. Calcium-dependent IP3 receptor dynamics

  29. PART 2:TECHNICAL PRACTICALITIES

  30. Regarding the necessity of keeping the compound in the dark. Storage. Vendor impurities - aftermarket purification. Cost control: recirculating and local perfusion. Handling caged compounds

  31. Caged glutamates: a consumer report Fastest: CNB- or desyl- Best optical cross-section: Brc- Most efficient two-photon effect: bis-CNB- Future potential for two-photon uncaging: Corrie’s Magickal Indoline Picking a caged compound

  32. Furuta et al. (1999) PNAS 96:1193

  33. Nd:YAG 355 nm

  34. Picking a light source Directing the light beam Spatial resolution How much light? Alignment and calibration Designing an uncaging setup

  35. If temporal only, light source can be uncollimated Flashlamps (Rapp) Mercury arc (Denk) Nd:YAG laser Argon laser Ti:S laser …see CSHL chapters by Delaney, Kandler Picking a light source

  36. Put as little UV-absorbing capacity as possible in the light path Capture the output Shuttering Directing the light to the specimen

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