Mitochondria are responsible for generating over 90% of the cell's ATP, are implicated in aging (the site of oxidative damage in cells), and are found to play a key role in apoptosis and calcium homeostasis. For many of these mitochondria! functions, there is only a partial understanding of the components involved, with even less information on mechanism and regulation. Almost all biochemical studies on mitochondria so far are based on bulk assays in which millions of mitochondria are isolated and chemical analyzed. Yet mitochondria are pleomorphic organelles with structural variations depending on the cell type, cell-cycle stage, intracellular metabolic state, and even its precise spatial position inside the cell. Within a given cell, therefore, there can be a high degree of variability between individual mitochondria in terms of morphology and membrane potential, and potentially also the biochemical composition. In diseases that trace themselves to mitochondrial malfunction, both normal and abnormal mitochondria are thought to co-exist in a given cell. Bulk assays that average over millions of mitochondria will necessarily mask their distinctness. Here we propose to develop a new method for the chemical analysis of single mitochondrion. There is a number of the advantages associated with our proposed method of single mitochondrial analysis: (1) Single-mitochondrion isolation and analysis from a cell is rapid, which is important for studying labile species in the mitochondrion, such as the reduced form of glutathione. (2) The ability to physically extract a mitochondrion minimizes potential issues of contamination, which are concerns in bulk mitochondrial preparations (e.g. with differential centrifugation) in which other subcelluar organelles (e.g. endoplasmic reticulum) can be present. (3) Single-mitochondrial analysis permits the study of the variation between mitochondria, a fact that is especially pertinent given the highly variable nature of mitochondria in a given cell (a situation termed heteroplasmy). (4) We can correlate directly biochemical information from a given mitochondrion with its morphology (e.g. size and shape), which is important in the context of apoptosis, because one morphological signature of apoptosis is mitochondrial fragmentation. (4) We can directly correlate the biochemical information from a given mitochondrion with its precise spatial location in the cell. A single cell has been observed (using microscopy) to contain different types of mitochondria; with our method we can study directly such variations to better understand the physiological origin of these differences. While our focus here is on the analysis of individual mitochondria, we believe our method can be applied equally well to the chemical analysis of other subcellular organelles and compartments.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB005197-03
Application #
7355970
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Zhang, Yantian
Project Start
2006-03-01
Project End
2010-02-28
Budget Start
2008-03-01
Budget End
2009-02-28
Support Year
3
Fiscal Year
2008
Total Cost
$316,803
Indirect Cost
Name
University of Washington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Cohen, Dawn E; Schneider, Thomas; Wang, Michelle et al. (2010) Self-digitization of sample volumes. Anal Chem 82:5707-17
Sgro, Allyson E; Chiu, Daniel T (2010) Droplet freezing, docking, and the exchange of immiscible phase and surfactant around frozen droplets. Lab Chip 10:1873-7
Han, Min-Joon; Chiu, Daniel T; Koc, Emine C (2010) Regulation of mitochondrial ribosomal protein S29 (MRPS29) expression by a 5'-upstream open reading frame. Mitochondrion 10:274-83
Hurtig, Johan; Chiu, Daniel T; Onfelt, Björn (2010) Intercellular nanotubes: insights from imaging studies and beyond. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2:260-76
Kuo, Jason S; Ng, Laiying; Yen, Gloria S et al. (2009) A new USP Class VI-compliant substrate for manufacturing disposable microfluidic devices. Lab Chip 9:870-6
Kuo, Jason S; Zhao, Yongxi; Ng, Laiying et al. (2009) Microfabricating high-aspect-ratio structures in polyurethane-methacrylate (PUMA) disposable microfluidic devices. Lab Chip 9:1951-6
Miller, Jennifer L; Cimen, Huseyin; Koc, Hasan et al. (2009) Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis. J Proteome Res 8:4789-98
Chiu, Daniel T; Lorenz, Robert M; Jeffries, Gavin D M (2009) Droplets for ultrasmall-volume analysis. Anal Chem 81:5111-8
Allen, Peter B; Doepker, Byron R; Chiu, Daniel T (2009) High-throughput capillary-electrophoresis analysis of the contents of a single mitochondria. Anal Chem 81:3784-91
Soung, George Y; Miller, Jennifer L; Koc, Hasan et al. (2009) Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomes. J Proteome Res 8:3390-402

Showing the most recent 10 out of 24 publications