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Imaging Ca2+ Signals in Xenopus Oocytes Sheila L. Dargan, Angelo Demuro, and Ian Parker

Summary

Xenopus oocytes have become a favored preparation in which to study the spatiotemporal dynamics of intracellular Ca2+ signaling. Advantages of the oocyte as a model cell system include its large size, lack of intracellular Ca2+ release channels other than the type 1 inositol trisphosphate receptor, and ease of expression of foreign receptors and channels. We describe the use of high-resolution fluorescence imaging techniques to visualize Ca2+ signals in Xenopus oocytes at levels ranging from global Ca2+ waves to single-channel Ca2+ microdomains.

Key Words: Ca2+; caged IP3; calcium; confocal; dye; flash photolysis; fluorescence; imaging; inositol; IP3; linescan; microinjection; microscopy; receptor; signaling; TIR; video rate; Xenopus oocytes.

1. Introduction

Xenopus oocytes are a favored model cell system in which to image Ca2+ signals evoked by inositol 1,4,5-trisphosphate (IP3) and possess distinct advantages over most other cell types. Intracellular Ca2+ liberation is mediated only by type 1 IP3 receptors in the absence of ryanodine receptors (1), their large size greatly facilitates intracellular injections, and they are among the best-characterized cells for Ca2+ signaling (2,3). Moreover, the ability of Xenopus oocytes to express foreign receptors and ion channels (4), such as calcium channels, further enhances the versatility of this already-favorable model system.

The protocols described in this chapter are aimed at the beginner in the field, assuming no previous knowledge of the methods. We describe the use of confocal microscopy techniques, together with flash photolysis of caged IP3, for imaging intra-cellular Ca2+ signals in Xenopus oocytes. We use "custom-built" confocal microscopes, but the methods are directly applicable to the use of commercial instruments. We also describe the use of total internal reflection fluorescence microscopy (TIRFM) for visualizing Ca2+ signals from individual voltage-gated channels.

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

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