Caged compounds: photorelease technology for control of cellular chemistry and physiology

Abstract

Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group (Fig. 1). Illumination results in a concentration jump of the biologically active molecule that can bind to its cellular receptor, switching on (or off) the targeted process. Virtually every kind of signaling molecule or second messenger, of every size| [mdash]|from protons to proteins| [mdash]|has been caged 1 .Why are caged compounds so useful? A single component of cellular chemistry can control the function of a cell, and such cellular regulation can be temporally or spatially defined, intracellular or extracellular, and amplitude-or frequency-modulated 2 . Photomanipulation of cellular chemistry using caged compounds provides a uniquely powerful means to interact with such cellular dynamics, as it can touch upon any one of the above dimensions. Thus, since light passes through cell membranes, uncaging can rapidly release a biomolecule in an intracellular compartment. This space is not readily accessible to many second messengers (for example, inositol-1,4,5-trisphosphate (IP 3 ), ATP, Ca 2+ , cAMP, cGMP) when they are applied to cells externally, as their charge makes them impermeable to the plasma membrane. Furthermore, uniform illumination results in release throughout the cytosol, or the release can be localized by focusing the uncaging beam on one part of a cell. Likewise, extracellular uncaging of neurotransmitters and hormones is tunable, allowing stimulation of many neurons simultaneously or of single synapses| [mdash]|by global or focused illumination, respectively. Light cannot only be directed, but also modulated in time andCorrespondence should be addressed to Graham C R Ellis-Davies (cagedca@hotmail.com). (Fig. 2) has been widely used to study many Ca 2+ -controlled processes. In particular, secretory processes in neuronal and non-neuronal cells have been extensively studied with caged calcium 22 . Rapid uncaging of glutamate using two-photon photolysis is particularly useful for the rational stimulation of visually identified synapses in complex tissue preparations such as acutely isolated brain slices from the hippocampus 23 (Fig. 2b). Finally, uncaging of mRNA in vivo in zebrafish is an excellen...

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