Capillary electrophoretic separation of nuclei released from single cells.
We report here the first capillary electrophoresis analysis of intact nuclei released on-column from single cells. Expression of the nuclear-targeted protein nuDsRed2 and the plasma membrane-bound farnesylated enhanced green fluorescent protein in cultured human DeltaH2-1 cells allowed fluorescent monitoring of the fate of these subcellular compartments upon injection of a single cell into the separation capillary. On-column treatment with digitonin allowed for the separation of the plasma membrane from the nucleus as indicated by their selective laser-induced fluorescence detection in two separate spectral regions. The data suggest that less than 0.1% of the plasma membrane remains bound to individually detected nuclei. In digitonin-treated cells, the electropherograms consisted of a prominent fluorescent peak attributed to nuDsRed2 localized to the nucleus and a collection of weakly fluorescent events (barely distinguishable from scattering) that seem to indicate additional localization of this protein to other subcellular regions. Taken together, this report points to the feasibility of studying intact organelles released from a single mammalian cell by capillary electrophoresis, which is a prerequisite to understanding the relevance of subcellular heterogeneity in biological systems.
Gunasekera
Nilhan
N
Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E., Minneapolis, MN 55455, USA.
Olson
Karen J
KJ
Musier-Forsyth
Karin
K
Arriaga
Edgar A
EA
D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000187:drug effects; D002467:Cell Nucleus; D002470:Cell Survival; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D019075:Electrophoresis, Capillary / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004072:Digitonin