Author:

Keywords:

0601 Biochemistry and Cell Biology, 1101 Medical Biochemistry and Metabolomics, Biochemistry & Molecular Biology, 3101 Biochemistry and cell biology

Abstract:

Measurement of free Ca2+ has established Ca2+ as the intracellular signal mediating the effects of agonists and pathogens in animal, plant and bacteria. But, prolonged elevation of cytosolic Ca2+ leads to cell injury or death. Ca2+binding proteins are found in sub-cellular compartments, e.g. the nucleus, ER, Golgi, vesicles, mitochondria, gap junctions and the plasma membrane. Two key questions now are: How does Ca2+ in these compartments control cell activity, defence and death? Are there mechanisms to target Ca2+ to specific sites within the cell, avoiding inappropriate activation elsewhere. We have developed a strategy to answer these questions by targeting aequorin and firefly luciferase to measure Ca2+ , ATP and protein modification at defined sites within the cell. A defective adenovirus expresses the indicators in a wide range of cell types. Efficiency of targeting was established by immunolocalisation, selective permeabilisation and GFP. Light signals can be imaged in single cells, and whole organs. Ca2+ and ATP have been correlated with Ca2+ binding protein gene expression and cell permeability. Free Ca2+ in the ER was 10-100 times that in the cytosol, i.e. l-l0μM. Raising cytosolic Ca2+, by release from the ER or using complement, highlighted a barrier to free Ca2+ between the nucleus and cytosol, necessary if Ca2+ is to be targeting independently to the nucleus. Agonist-stimulated Ca2+ signals have been detected in the nucleus, ER and cytosol. Release of Ca2+ from the ER activates plasma membrane channels, but the fractional release of ER Ca2+ is small. Ca2+ stress activates the gene for calreticulin and other chaperones. Calreticulin may also be targeted to the nucleus. Imaging of Ca2+ and ATP in single cells showed that when the plasma membrane is attacked the rise in cytosolic Ca2+ precedes a decrease in ATP and permeabilisation to small molecules A key question now is whether this decrease in ATP is responsible for the cell crossing the ultimate Rubicon, lysis. Genetic engineering of bioluminescent proteins, combined with fluors and live cell imaging opens up a new era for investigating how Ca2+ can act at discrete sites within individual cells and intact organs.