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56 Cell-Penetrating Peptides: Processes and Applications
FIGURE 3.1 (Color Figure 3.1 follows p. 14.) Confocal microphotographs showing internalization
of biotinyl-transportan. Bowes cells were incubated with 10 µM biotinyl-transportan
at 0˚C (A to C) or at 37˚C (D to F) for 60 min. Transportan was visualized by streptavidin-
Texas Red (A, D) and membranes were stained with concanavalin A-FITC (B, E); the
combined image is shown on C, F. (Reprinted from Pooga, M. et al., FASEB J., 12, 67, 1998.
With permission.)
However, there is one principal difference in respective cellular localization: after
1 h incubation at 0°C, the peptide does not accumulate in the nuclei. The reason for
this is not clear yet, but could be explained by a slower redistribution of the peptide in
the cell interior or a lower degree of intranuclear accumulation giving a concentration
under the detection limit for this method. In addition, degradation of transportan in the
cells at 37°C, but not at 0°C, can lead to formation of fragments capable of nuclear
translocation. However, the analysis of internalized transportan by electrophoresis indicated
the presence of intact transportan, but not the shorter fragments, which makes
nuclear translocation after partial degradation of transportan less probable.
The nature of the cytosolic structures in which transportan concentrates was
assessed using the fluorescently labeled lectin concanavalin A (Con A). Con A binds
to glycoproteins with high mannose content and also stains, along with the plasma
membrane and nuclear envelope, different intracellular membranous structures, and