Analysis of mitochondria isolated from single cells

Abstract

Bulk studies are not suitable to describe and study cell-to-cell variation, which is of high importance in biological processes such as embryogenesis, tissue differentiation, and disease. Previously, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to measure the properties of organelles isolated from millions of cells. As such, these bulk measurements reported average properties for the organelles of cell populations. Similar measurements for organelles released from single cells would be highly relevant to describe the subcellular variations among cells. Toward this goal, here we introduce an approach to analyze the mitochondria released from single mammalian cells. Osteosarcoma 143B cells are labeled with either the fluorescent mitochondrion-specific 10-N-nonyl acridine orange (NAO) or via expression of the fluorescent protein DsRed2. Subsequently, a single cell is introduced into the CE-LIF capillary where the organelles are released by a combined treatment of digitonin and trypsin. After this treatment, an electric field is applied and the released organelles electromigrate toward the LIF detector. From an electropherogram, the number of detected events per cell, their individual electrophoretic mobilities, and their individual fluorescence intensities are calculated. The results obtained from DsRed2 labeling, which is retained in intact mitochondria, and NAO labeling, which labels all mitochondria, are the basis for discussion of the strengths and limitations of this single-cell approach.

Electrochemical Probes for Detection and Analysis of Exocytosis and Vesicles

“…[51] Artificial vesicles were encapsulated with a fluorescent dye, and those vesicles containing the fluorophore elicited a signal in the resultant electropherogram, whereas a control suspension was rendered transparent to the detection scheme. This group has had success in applying the CE-LIF methodology to investigate a number of subcellular components including cell nuclei, [52] mitochondria, [53] and various acidic organelles. [54] A long-standing assumption in the study of exocytosis is that release is quantal in nature and that each quantum is comprised of the contents of a single vesicle.…”

Recent advances in the analysis of biological particles by capillary electrophoresis

“…A general review covering the effort to develop techniques for the single-cell analysis of individual organelles has appeared [38]. The CE analysis of subcellular particles has included the analysis of new types of subcellular particles [7,39,40], separation conditions [41], improved detection arrangements [42,43], new applications [44][45][46] and new sampling and labeling schemes [47][48][49].…”

CE analysis of the acidic organelles of a single cell

“…The permeabilized membrane allowed the trypin to access the subcellular environment where it proteolyzed structural proteins (e.g., tubulin [39]), further aiding in the release of organelles that had been anchored to the cytoskeleton [38]. That digitonin and trypin can disrupt the plasma membrane, thereby releasing the organelles, has been illustrated previously [19]. After 3 min incubation, CE separations were performed at 2300 V/cm in CE buffer.…”

“…In regard to the subcellular analysis of single cells, CE has been used to sample small subcellular regions, and to measure properties of the whole nucleus of a single cell [17,18]. The ability to detect individual mitochondrial particles by CE-LIF after their release from single cells has been recently described [19]. This report shows that the number of mitochondria per cell, the cardiolipin content per mitochondrion, the DsRed2 (red fluorescent protein) content per mitochondrion, and the electrophoretic mobilities for the mitochondria of the same cell are all heterogeneous.…”