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Model Amphipathic Peptides 81
relative fluorescence
160
140
120
100
80
60
40
20
0
0 5 10 15 20 25 30 35
FIGURE 4.6 Time course of the fluorescence intensity within the cytosol after exposure of
LKB Ez7 cells at 37°C to 1 µM I, VI, IX, or X, respectively, normalized to the fluorescence
intensity of the external peptide solution at time point 0.
4.3.3 MONITORING OF CELLULAR UPTAKE OF MAPS BY AN ON-LINE CLSM
PROTOCOL THAT AVOIDS BIAS OF RESULTS BY WASH-OUT
min
The notion that nonamphipathic peptides of the MAP series are also able to penetrate
mammalian plasma membranes could be confirmed by using an on-line CLSM
protocol for monitoring cellular uptake. This protocol enabled measurement of the
intracellular fluorescence in the presence of external fluorescent substrate, 30,31 thus
avoiding bias of the results by wash-out. The correction of the measured intracellular
fluorescence by out-of-focus contributions of the extracellular fluorescence was
achieved in this protocol by means of the function of the axial response of the
microscope, which was itself determined using the cell-impermeant dye carboxyfluorescein.
31 These on-line CLSM studies revealed an extensive and very rapid
translocation of both amphipathic and non-amphipathic I-related peptides across
mammalian plasma membranes proceeding to a comparable extent within a few
minutes (Figure 4.6; see Reference 32).
In most cases, the fluorescence appeared evenly distributed throughout the cytosol
and nucleus, accompanied by a perinuclear punctuate pattern, suggesting that
nonendocytic as well as endocytic modes of uptake are involved. The predominance
of nonendocytic processes was indicated by the fact that exposure of the cells at
0°C to the peptides also led to extensive intracellular fluorescence, with the signals
not significantly different from those at 37°C. 32
A different fluorescence pattern, however, now exhibiting analogy to the results
obtained by the HPLC protocol (Figure 4.7), was observed after multiple cold
washing of the peptide-exposed cells. 32 In washed cells preloaded with peptides of
I
VI
IX
X