Mitochondria are the central regulator of apoptosis, a process initiated by the activation of the mitochondrial permeability transition pore (mtPTP), an aggregate of several mitochondrial proteins. When this pore opens, the critical membrane polarization of the mitochondrial inner membrane disappears and ions equilibrate between the matrix and cytosol resulting in mitochondrial swelling. This leads to release of the contents of the mitochondrial intermembrane space into the cell cytosol, including a number of cell death promoting factors killing the cell. The mtPTP can be activated by uptake of excessive Ca++;increased oxidative stress;decreased mitochondrial membrane potential, and reduced ADP and ATP. It is generally agreed upon that repression of apoptosis is one of the fundamental steps in tumorigenesis. Cancer cells acquire unresponsiveness to apoptosis facilitating signals, thus enabling uncontrolled proliferation. For this reason, the induction of apoptosis is one of the modes of actions of chemotherapeutic compounds. In order to allow further high throughput studies of the biochemical facilitators and inhibitors, of apoptosis, and to determine if changes in individual mitochondrial membrane potential P are important to cellular metabolism, we need to develop a system to monitor P in individual mitochondria. To accomplish this objective, we propose to extend studies that have monitored the action potentials in neurons using an array of parallel electrodes to which the mitochondria are adhered. Our thesis is that a nanoelectrode technology can be developed to capacitively measure membrane potential across the mitochondrial inner membrane phospholipid bilayer without actually penetrating the membrane. We propose to develop nano-electrical transduction sensor arrays with sufficiently high spatial and temporal resolution to monitor the charge changes on the surface of a mitochondrion sized lipid vesicle and the individual mitoplast. With this technology, we will then interrogate the regulation of P in normal and cancer cells. Several key features on mitochondrial metabolism are now recognized as important to the alteration of cancer cell mitochondrial function: changes in the Akt signal transduction pathway, induction of hexokinase II, alteration an adenine nucleotide translocator (ANT) isoform expression, down regulation of the SOC2 cytochrome c oxidase (complex IV, COX) assembly factor, mutation in mitochondrial DNA (mtDNA) genes, and modulation of the mitochondrial permeability transition pore (mtPTP) and its interaction with the pro- and anti- apoptotic Bcl2 family proteins. While all of these are important factors in the alteration of cancer cell metabolism, they still fall short of explaining the near universal alterations in mitochondrial function observed in cancer cells. A high throughput technology to monitor P in mitochondria will allow further studies of these issues in cancer biology.
In order to allow further high throughput studies of the biochemical facilitators and inhibitors, of apoptosis, and to determine if changes in individual mitochondrial membrane potential ?P are important to cellular metabolism, we need to develop a system to monitor ?P in individual mitochondria. We propose to develop nano-electrical transduction sensor arrays. With this technology, we will then interrogate the regulation of ?P in normal and cancer cells. A high throughput technology to monitor ?P in mitochondria will allow further studies in cancer biology that explain the near universal alterations in mitochondrial function observed in cancer cells.
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