tailieunhanh - Báo cáo khoa học: Detecting mitochondrial permeability transition by confocal imaging of intact cells pinocytically loaded with calcein

When studiedin vitro, mitochondrial permeability transition (MPT) is associated with an increase in mitochondrial per-meability to solutes up to 1500 Da in mass and a loss of electrical potential difference across the inner mitochondrial membrane (Dwmit). The MPT has been implicated as being important in cellular calcium homeostasis, autophagy and cell death via necrosis and apoptosis. | Eur. J. Biochem. 269 3990-3997 2002 FEBS 2002 doi Detecting mitochondrial permeability transition by confocal imaging of intact cells pinocytically loaded with calcein Rachel A. Jones Alison Smail and Mark R. Wilson Department of Biological Sciences University of Wollongong New South Wales Australia When studied in vitro mitochondrial permeability transition MPT is associated with an increase in mitochondrial permeability to solutes up to 1500 Da in mass and a loss of electrical potential difference across the inner mitochondrial membrane AWmit . The MPT has been implicated as being important in cellular calcium homeostasis autophagy and cell death via necrosis and apoptosis. Thus it is important to develop a valid technique for accurate measurement of this phenomenon in intact cells. We developed a procedure for the detection of MPT in intact cells that avoids the disadvantages associated with earlier approaches. In this new technique unmodified green-fluorescent calcein is simul taneously introduced into the cytosol of millions of cells by the process of pinocytic loading and to identify the position of individual mitochondria and to measure AWmit the cells are counter-stained with a red-fluorescing potentiometric dye. Using this approach with a variety of cell types we demonstrate that cytosolic calcein is excluded from normal polarized mitochondria but enters them during MPT. This technique may be valuable in studies investigating the cellular functions of MPT. Keywords calcein confocal microscopy mitochondria permeability transition pinocytic loading. The mitochondrial permeability transition MPT has been studied in isolated mitochondria for decades 1 . Classically when studied in vitro the phenomenon is associated with loss of electrical potential difference across the inner mitochondrial membrane AWmit and mitochondrial swelling resulting from an increase in the permeability of mitochondria to solutes up to 1500 Da in mass 1