In the proposed studies, we aim to develop and validate technology to allow for high-throughput assays of inhibitors and inducers of mitochondrial depolarization, the point of no return for apoptosis. As apoptosis is a key unregulated pathway in tumor progression, its study and ultimately control is of paramount important in cancer biology. Preliminary studies by some of us have shown a correlation between patient cancer progression-free survival rate and the response of mitochondria (depolarization of the inner membrane, permeabilization of the outer membrane) to pro-apoptotic BH3 peptides in digitonin permeabilized patient primary tumor cells. We also have shown that the patients whose mitochondria are more easily depolarized with BH3 peptides respond better to chemotherapy. In proof of concept work, we demonstrated an on-chip nanofluidic technology that was able to trap individual, living mitochondria isolated from cell and tissues, and to interrogate the membrane potential using potential sensitive fluorescence probes of these individual mitochondria in response various chemical environments, including substrates and inhibitors of the electron transport chain, as well as calcium challenges which resulted in mitochondrial flickering and depolarization. In addition, our initial work demonstrated in preliminary studies the electrical sensing of the opening and closing of individual ion channels in lipid bilayers using nanotube electrodes. In the proposed studies we will develop and validate this technology to sense the opening and closing of individual ion channels in mitoplasts and mitochondria, study their statistics and timing, and to develop a platform to enable, ultimately, high throughput assays of the electrophysiology of the mitochondrial electron transport chain and membrane depolarization at the single ion channel level. The technology will be transformative in three ways: First, it will validate a qualitatively new assay for study of apoptosis. Deregulation of apoptosis is well known as one of 6 hallmarks of cancer, however, methods to study mitochondrial depolarization have been lacking. Second, it will allow for a qualitatively new way to study electrophysiology at the single ion channel level. This will allow unprecedented studies of timing, location, and statistics of the mitochondrial membrane flickering and depolarization. Finally, the overall technology will enable new studies of mitochondrial metabolism. Cancer metabolism is one of the 2 new hallmarks added last year to cancer, and this instrumentation will enable the understanding of one important component of metabolic flux (namely, membrane potential). Because our high throughput technology will enable assays of thousands of individual mitochondria from small numbers of cells (even a single cell), it will enable an advance in instrumentation to study the interrelationships between metabolism, stem cells, and cancer biology.

Public Health Relevance

This project develops techniques to study programmed cell death and its regulation by proteins and molecules. Because regulating this is important to understand cell growth and proliferation, it may aid to understand how this process goes wrong in cancer (uncontrolled cell growth and proliferation), and eventually enable new therapies to treat cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants Phase II (R33)
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Special Emphasis Panel (ZCA1-SRLB-Q (O2))
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Knowlton, John R
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University of California Irvine
Engineering (All Types)
Schools of Engineering
United States
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