In Vivo Measurements of Cytosolic Calcium in Dictyostelium discoideum

Summary

The involvement of calcium signalling during chemotaxis in Dictyostelium discoideum is well documented. Spatiotemporal increases of intracellular calcium ([Ca²⁺]_i) have been observed within seconds of stimulation with the chemoattractants folic acid and cAMP. This rise in [Ca²⁺]_i localises to the rear cortex of the cell (J. Cell Sci. 109:2673–2678, 1996) and has been found to be not essential for chemotaxis, but likely to be involved in fine tuning of chemotactic responses (EMBO J. 19:4846–4854, 2000). Measurements of cytosolic Ca²⁺ ([Ca²⁺]_c) responses have involved the use of different Ca²⁺ probes including ectopically expressed aequorin (a Ca²⁺-sensitive photoprotein), the fluorescent dye fura-2-dextran and the radioisotope ⁴⁵Ca²⁺. The aequorin method (J. Cell Sci. 110:2845–2853, 1997) offers nonperturbing, real-time measurement of cytosolic free Ca²⁺ in suspensions of cells, but the low levels of light emission preclude measurements on individual cells. Fura-2 imaging (Cell Calcium 22:65–74, 1997; Eur. J. Cell Biol. 58:172–181, 1992; Biochem. J. 332:541–548, 1998; BMC Cell Biol. 6:13, 2005) has the advantage of allowing Ca²⁺ responses to be observed in individual cells so that the subcellular localisation of the response and differences amongst individual cells can be observed. However data collection is more labour intensive, much smaller numbers of cells are sampled, the cells are unavoidably damaged physically during loading and the time resolution (s) is much less than that provided by the aequorin method (ms). ⁴⁵Ca²⁺ uptake assays (Cell Biol. Int. Rep. 2:71–79, 1978; J. Cell Biol. 112:103–110, 1991) allow measurement of Ca²⁺ influx from the medium by cell suspensions with a time resolution of the order of seconds. Radioactive Ca²⁺ uptake measurements are unsullied by but equally do not provide information about Ca²⁺ efflux, intracellular Ca²⁺ release or sequestration or changes in cytosolic free Ca²⁺ levels.