Mitochondria are well-characterized regarding their function in both energy production and regulation of cell death; however, the heterogeneity that exists within mitochondrial populations is poorly understood. Typically analyzed as pooled samples comprised of millions of individual mitochondria, there is little information regarding potentially different functionality across subpopulations of mitochondria. Herein we present a new methodology to analyze mitochondria as individual components of a complex and heterogeneous network, using a nanoscale and multi–parametric flow cytometry-based platform. We validate the platform using multiple downstream assays, including electron microscopy, ATP generation, quantitative mass-spectrometry proteomic profiling, and mtDNA analysis at the level of single organelles. These strategies allow robust analysis and isolation of mitochondrial subpopulations to more broadly elucidate the underlying complexities of mitochondria as these organelles function collectively within a cell.
Ciona intestinalis, a common sea squirt, exhibits lower reproductive success at the upper extreme of the water temperatures it experiences in coastal New England. In order to understand the changes in protein expression associated with elevated temperatures, and possible response to global temperature change, we reared C. intestinalis from embryos to adults at 18°C (a temperature at which they reproduce normally at our collection site in Rhode Island) and 22°C (the upper end of the local temperature range). We then dissected ovaries from animals at each temperature, extracted protein, and measured proteomic levels using shotgun mass spectrometry (LC-MS/MS). 1532 proteins were detected at a 1% false discovery rate present in both temperature groups by our LC-MS/MS method. 62 of those proteins are considered up- or down-regulated according to our statistical criteria. Principal component analysis shows a clear distinction in protein expression pattern between the control (18°C) group and high temperature (22°C) group. Similar to previous studies, cytoskeletal and chaperone proteins are upregulated in the high temperature group. Unexpectedly, we find evidence that proteolysis is downregulated at the higher temperature. We propose a working model for the high temperature response in C. intestinalis ovaries whereby increased temperature induces upregulation of signal transduction pathways involving PTPN11 and CrkL, and activating coordinated changes in the proteome especially in large lipid transport proteins, cellular stress responses, cytoskeleton, and downregulation of energy metabolism.
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