Results
Fluorescence labeling
reveals cellular uptake and delayed endo-lysosomal escape of penetratin
Penetratin was labeled with two different fluorescent dyes in order to
study the mechanism and kinetics of its cellular uptake. The
fluorescence of naphthofluorescein (NF) is quenched at acidic pH
enabling studying the release of penetratin from the acidic
endo-lysosomal compartment (Qian, Dougherty & Pei, 2015). In order to
complement the information on endo-lysosomal escape we chose labeling
with AFDye532, a dye exhibiting pH-independent fluorescence to report on
the total cellular content of penetratin. The cellular fluorescence of
AFDye532-penetratin is proportional to the total cellular uptake of
penetratin, the intensity of NF-penetratin characterizes its
concentration in non-acidic compartments (mainly the cytosol), whereas
the ratio of NF-labeled and AFDye532-labeled penetratin intensities
reveals the fractional escape of the cell-penetrating peptide from
acidic compartments. The molecular weight and purity of labeled
penetratin was checked by mass spectrometry and high performance liquid
chromatography (Suppl. Fig. 1). We developed an approach based on flow
cytometric measurement of cell-associated fluorescence signals to
separately measure the kinetics of cellular uptake and endo-lysosomal
release of these fluorescently labeled penetratins. Cells were incubated
in the continuous presence of an equimolar mixture of NF-penetratin and
AFDye532-penetratin (5 µM of each) at 37°C, and the fluorescence
intensity of cells was measured in a time-correlated manner. After
removing cells with compromised membrane permeability based on DAPI
staining from the dataset and compensating for spectral crosstalk, the
time-dependent change of fluorescence intensity was plotted using moving
average smoothing. The signal of AFDye532-penetratin, corresponding to
the total cellular content of penetratin, reached saturation at 200-400
seconds in two different cell lines, while a significantly delayed
saturation of penetratin concentration in the neutral, cytoplasmic
compartment at 800-1000 seconds was observed based on the pH-sensitive
fluorescence of NF-penetratin (Fig. 1). The ratio of NF to AFDye532
intensities, characterizing the fraction of penetratin in neutral
compartments, initially declined, corresponding to a preferential
presence of penetratin in acidic endosomes, followed by a gradual
increase reaching saturation at approximately 800-1000 seconds (Fig. 1).
Since flow cytometry lacks subcellular resolution, we correlated the
fluorescence intensities of the two reporters with their cellular
location. Confocal microscopic investigation of the distribution of the
fluorescent penetratin derivatives revealed that AFDye532-penetratin
exhibits bright fluorescence even in endosomes where the fluorescence of
NF is quenched and that the cell-associated fluorescence of NF
originates from outside the endo-lysosomal compartment (Fig. 2). This
observation confirms that interpretation of the fluorescence intensity
of AFDye532-penetratin and NF-penetratin, measured by flow cytometry, as
total cellular penetratin uptake and the amount outside endosomes,
respectively, is indeed correct. Since AFDye532 and NF form a Förster
resonance energy transfer pair, anticorrelation between their
fluorescence intensities could also have been caused by energy transfer,
i.e. a high local concentration of the acceptor (NF) quenching the
fluorescence of the donor (AFDye532). In order to exclude this
possibility, the equimolar mixture of fluorescent penetratins was
supplemented with 10 µM unlabeled penetratin. If energy transfer is to
blame for the anti-correlation between the fluorescence intensities of
the two dyes, dilution of their local concentration with unlabeled
penetratin is expected to eliminate or reduce this anti-correlation.
However, the time dependence of the fluorescence intensities and their
ratio were not changed significantly by the presence of unlabeled
penetratin allowing us to conclude that the fluorescence intensities of
AFDye532 and NF correctly report total cellular uptake of penetratin and
its concentration in neutral compartments, respectively (Suppl. Fig. 2).
In summary, we established a flow cytometric approach revealing the
different kinetics of cellular uptake and endo-lysosomal release of
penetratin setting the stage for further analysis of the effect of
dipole potential-modifying agents on penetratin uptake.