Intracellular Concentration Measurements in Adherent Cells: A Comparison of Import Efficiencies of Cell-Permeable Peptides

Abstract

A protocol was developed for performing intracellular concentration measurements in flat adherent tissue culture cells by fluorescence correlation microscopy (FCM). Determination of the number of molecules in the confocal detection volume had to account for background fluorescence caused by molecules adsorbed to the surface of the measurement chamber. Such a background signal leads to a decrease in the amplitude of the autocorrelation function, and thereby to the calculation of an erroneously high number of molecules. Because of the spatial heterogeneity of the background intensity, a method was devised by which its contribution to the total fluorescence could be determined directly from each individual autocorrelation measurement. This method was applied to a comparison of the import efficiencies of different cellpermeable peptides at nanomolar concentrations. The Antennapedia homeodomainderived peptide penetratin was imported about three times as efficient as the basic fibroblast growth factorderived MTS peptide. Both peptides equilibrated between cytoplasm and nucleus. A relatively high mobility of these molecules inside the cells indicated that they may be rapidly degraded by cytosolic proteases. Based on these results, it will be possible to determine intracellular concentrations of inhibitors linked to import peptides directly by FCM at nanomolar concentrations and to optimise such constructs for proteolytic stability.