Calcium is an important signal to different cellular functions and to the apoptotic signaling (Smaili et al, 2003). Calcium homeostasis is maintained by pumps, transporter and proteins that bind to the ion. It is also stored in different intracellular compartments as endoplasmic reticulum, mitochondria, golgi among others. Alterations in the calcium homeostasis and intracellular stores may control apoptotic cell death (Rizzuto and Pozan, 2006). Apoptosis is regulated by several different factors as the Bcl-2 family of proteins which is composed by pro-apoptotic members (e.g. Bax, Bad and Bid) and anti-apoptotic members (e.g. Bcl-2 and Bcl-xL) (Smaili et al., 2003). Bax is predominantly found in the cytosol as a monomeric soluble protein and, in lower levels, in the mitochondrial membranes. Under apoptotic stimuli there is a conformational change in the protein which induces its translocation to mitochondria and endoplasmic reticulum (Antonsson, 2004). Studies in our laboratory have shown that during Bax translocation there is an increase in cytosolic calcium released from intracellular stores. In addition, agonists that mobilize these stores also induce Bax translocation. To clarify the mechanisms involved in the relationship between the calcium mobilization and Bax translocation, it is important to find the right tools to measure the source of the intracellular calcium released (e.g. mitochondria, endoplasmic reticulum and nuclei) during translocation. Therefore, the objective of this study is to establish cell lines with permanently expressing EGFP-Bax, DsRed-Bax, EGFP-Bcl-xL and mitochondrial and nuclei Erica. These cells will allow us to measure calcium and translocation of Bax and Bcl-xL simultaneously using high resolution fluorescence microscopy. The transfections will be performed in immortalized astrocytes, which will be also developed in this project, and in the MCF-7 (human breast adenocarcinoma cell line) cell line.
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