A changed cameleon

Ca²⁺ is one of the most versatile signalling factors that has been identified so far, and the release of Ca²⁺ from the endoplasmic reticulum (ER) — which functions as an intracellular Ca²⁺ storehouse — has crucial roles in the regulation of processes such as apoptosis and exocytosis. In Proceedings of the National Academy of Sciences, Roger Tsien, John Reed and colleagues now describe the development of an improved genetically encoded fluorescent sensor that can monitor Ca²⁺-concentration fluctuations in the ER ([Ca²⁺]_ER), and they have used this sensor to study the role of the anti-apoptotic protein B-cell lymphoma-2 (Bcl2) in breast cancer cells.

Amy Palmer in the Tsien laboratory started with the original cameleon construct — two fluorescent proteins (cyan fluorescent protein (CFP) and citrine) separated by calmodulin (CaM) and a CaM-binding peptide. In the presence of Ca²⁺, CaM interacts with the CaM-binding peptide, and CFP emission decreases as citrine emission increases, which is indicative of increased fluorescence resonance energy transfer (FRET). Their aim was to change this cameleon to decrease its perturbation by endogenous CaM and to vary its Ca²⁺ affinity.

Using previously obtained structural data, Palmer targeted salt-bridge interactions between CaM and the CaM-binding peptide. Compared to the wild-type peptide, a mutant peptide with four charge reversals had a 10,000-fold lower affinity for wild-type CaM. Palmer then made compensatory charge reversals in CaM with the aim of restoring its affinity for the mutant peptide. She named the mutant CaM and peptide pair Design 1 (D1), and cloned it between CFP and citrine to produce an altered cameleon.

Next, Palmer studied the properties of the D1 cameleon. In the presence of Ca²⁺, the magnitude of the observed FRET changes was comparable to that seen for the original cameleon. In addition, the Ca²⁺ titration curve for the D1 cameleon showed that it is ideal for monitoring [Ca²⁺]_ER (that is, Ca²⁺ concentrations in the low micromolar to hundreds of micromolar range). Furthermore, the D1 cameleon is not perturbed by large excesses of endogenous CaM, and it has a faster k_off than previous cameleons, so it can monitor rapidly changing Ca²⁺ dynamics.

The addition of an ER signal sequence and ER-retention tag resulted in the D1 cameleon being specifically localized to the ER, and Palmer showed that the D1 cameleon can detect both increases and decreases in [Ca²⁺]_ER with a much better sensitivity than previous cameleons. Furthermore, she showed that the D1 cameleon and the fluorescent dye fura-2 can be used together to allow the simultaneous monitoring of [Ca²⁺]_ER and [Ca²⁺]_cytosol, respectively.

Moving to a biological application for the improved cameleon, Palmer collaborated with Can Jin in the Reed laboratory to study the relationship between Bcl2, [Ca²⁺]_ER and apoptosis in a breast cancer cell line. Overexpressing Bcl2 in these cells, which is known to make them resistant to apoptosis, lowered [Ca²⁺]_ER under resting conditions by increasing Ca²⁺ leakage and altered the Ca²⁺ oscillations that were induced by ATP. In addition, the authors showed that a green-tea compound, epigallocatechin gallate, induced apoptosis in Bcl2-overexpressing breast cancer cells in a dose-dependent manner. Epigallocatechin gallate binds to Bcl2 and increases [Ca²⁺]_ER by blocking Bcl2-mediated leakage. They are now trying to elucidate the exact link between Bcl2 inhibition, increased [Ca²⁺]_ER and apoptosis.