Abstract
The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which approximately 40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.
Highlights
Many cell functions depend on a polarized, non-equilibrium distribution of molecular constituents
To establish the accuracy of the confocal laser scanning microscope (CLSM) for quantifying enhanced green fluorescent protein (EGFP) expression in living cells, we measured the fluorescence of a line of CHO cells that express EGFP, and compared the EGFP mass per cell estimated from CLSM data with the mass estimated by western blotting and fluorimetry
We determined the amount of EGFP in each voxel and summed the amounts over the cell volume to estimate the total mass of EGFP per cell
Summary
Many cell functions depend on a polarized, non-equilibrium distribution of molecular constituents. Photoreceptors provide an excellent example in that both soluble and membraneassociated components of phototransduction function in a cellular compartment distinct from the site of macromolecular synthesis. Efforts to characterize the cytoplasmic space with fluorescent molecular probes of various sizes have revealed a sizedependent partitioning into different subcellular regions (Janson et al, 1996; Luby-Phelps, 2000). The most likely explanation of this partitioning is ‘sieving’, i.e. the existence of compartments to which access depends on particle size. Quantification of the cytoplasmic space accessible to macromolecules of varying sizes is critical for defining the equilibrium distribution of a soluble macromolecule in a cell, providing a baseline against which nonequilibrium can be gauged
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