Abstract
Calcium handling by mitochondria is important both because mitochondria can shape the cytosolic Ca2+ signals and because changes in mitochondrial Ca2+ concentration ([Ca2+]M) are important for controlling physiological functions such as respiration or programmed cell death. Accurate measurements of [Ca2+]M require selective location of the Ca2+ probe inside mitochondria and this is best achieved by targeting protein probes to the mitochondrial matrix. Aequorins are very adequate as Ca2+probes because: (1) they allow molecular engineering for targeting or for changing the Ca2+ affinity; (2) do not require irradiation for measurements; (3) Ca2+ buffering is small; (4) have a very steep Ca2+-dependence and a very wide dynamic range, which makes them ideal for detecting and quantifying Ca2+ microdomains. Consumption and low light output are some of its drawbacks that make calcium imaging a hard task. Here, we describe a procedure that overcomes these disadvantages by combining herpes simplex virus type 1(HSV-1)-based expression of targeted aequorins with photon-counting imaging. This methodology allows real-time resolution of changes of [Ca2+]M by photon counting imaging at the single-cell level. Since HSV virus is neurotrophic, the method is adequate for measuring [Ca2+]M in living neurons.
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Acknowledgments
Financial support from the Spanish Ministerio de Educación y Ciencia (grants BFU2007-60157, BFU2005-02078 and BFU2006-05202), Instituto de Salud Carlos III (PI07/0766) and the Junta de Castilla y León (VA-088/A06) is gratefully acknowledged.
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Núñez, L., Villalobos, C., Alonso, M.T., García-Sancho, J. (2010). Ca2+ Imaging of Intracellular Organelles: Mitochondria. In: Verkhratsky, A., Petersen, O. (eds) Calcium Measurement Methods. Neuromethods, vol 43. Humana Press. https://doi.org/10.1007/978-1-60761-476-0_9
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