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Analysis of subcellular structures isolated from single cells

Recent Research from the group of Professor Edgar Arriaga.

In order to maintain cellular function, subcellular structures (organelles) have a complex organization and a wide range of properties even for organelles of the same type. In particular, the architecture of mitochondria varies from a tight network to hundreds of independent organelles, which collectively fulfill mitochondrial roles (e.g. ATP production, lipid metabolism, and calcium homeostasis) in the cell. Because organelle-to-organelle variations are of high importance in biological processes such as embryogenesis, tissue differentiation, and disease, there is great demand to develop highly sensitive techniques to investigate the properties of organelle ensembles in single cells. In the January issue of Analytical and Bioanalytical Chemistry (Anal. Bioanal. Chem. 387, 107-118, 2007), Ryan D. Johnson and co-workers in the Arriaga group reported the first analysis of mitochondria isolated from single 143B human osteosarcoma cells. These cells were labeled via expression of the fluorescent protein DsRed2, targeted to mitochondria (Figure 1A). Subsequently, a single cell was introduced into the lumen of a capillary where the organelles are released by a combined treatment of digitonin and trypsin (Figure 1B). After this treatment, an electric field was applied to cause electromigration of the released organelles toward a laser-induced fluorescence detector (Figure 1C). From an electropherogram (Figure 1D), the number of detected events per cell, their individual electrophoretic mobilities (Figure 1F), and their individual fluorescence intensities (Figure 1E) are calculated. The Arriaga group is currently working toward extending this new bioanalytical technology to investigate how giant and normal mitochondria within the same cell contribute to the aging process and how acidic organelles contribute to drug resistance.

Figure 1. Analysis of organelles taken from single cells. (A) A single 143B cell expressing DsRed2, targeted to mitochondria. (B) One cell is introduced into a fused silica capillary and placed between two plugs of buffer capable of disrupting the plasma membrane and the cytoskeletal network (1), the cell is electrokinetically displaced (2), and upon plasma membrane disruption, organelles are released (3). (C) Detail of a post-column laser-induced fluorescence detector. (D) Electropherogram displaying detection of the individual organelles from a single cell. (E) Intensities and number of organelles taken from three single cells. (F) Average of electrophoretic mobility distributions of organelles taken from three single cells.

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