Quantitative proteomics reveals oxygen-dependent changes in neuronal mitochondria affecting function and sensitivity to rotenone.

Mitochondria are implicated in a variety of degenerative disorders and aging. Mitochondria are responsive to the oxygen in their environment, yet tissue culture is performed at atmospheric (21%) oxygen and not at physiological (1-11%) oxygen levels found in tissues. We employed imaging of mitochondrial probes, mass spectrometry, Western blots, and ATP assays of the human neuroblastoma cell-line SH-SY5Y and imaging of mitochondrial probes in human primary neurons under standard nonphysiological oxygen conditions (atmospheric) and under physiological oxygen levels in the nervous system to assess the impact of oxygen on mitochondrial function.

Metabolic imaging of the organ of corti--a window on cochlea bioenergetics.

Hair cell loss is a major cause of sensorineural hearing loss. We have developed a method to examine metabolic events in hair cells in response to stimuli known to cause hair cell loss, such as acoustic trauma and aminoglycoside administration. The method employs two-photon excitation of the metabolic intermediate, reduced nicotinamide adenine dinucleotide (NADH), in hair cell mitochondria in an explanted mouse cochlea.

Fluorescence microscopy methods in the study of protein structure and function.

As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we describe two fluorescence microscopic methodologies applied to the outer hair cell-specific membrane protein prestin: the intensity and fluorescence lifetime (FLIM) variants of FRET (Fluorescence Resonance Energy Transfer), used in the study of protein-protein interactions, and the Scanning Cysteine Accessibility Method (SCAM), used for the determination of protein conformation.

Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy.

Currently there is no accepted method to measure the metabolic status of the organ of Corti. Since metabolism and mitochondrial dysfunction are expected to play a role in many different hearing disorders, here for the first time we employ two-photon metabolic imaging to assess the metabolic status of the cochlea. When excited with ultrashort pulses of 740-nm light, both inner and outer hair cells in isolated murine cochlear preparations exhibited intrinsic fluorescence.

Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy.

Endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides an intrinsic indicator of the cellular metabolic state, but prolonged monitoring is limited by photobleaching and/or phototoxicity. Multiphoton excitation of NADH by ultrashort, 740-nm laser pulses provides a significant improvement over UV excitation by eliminating peripheral photobleaching; however, molecules within the subfemtoliter excitation volume remain susceptible. We have investigated the photophysical mechanisms responsible for multiphoton photobleaching of NADH in living cells to permit the imaging technique to be optimized.