Fluorescein redirects a ruthenium-octaarginine conjugate to the nucleus

Abstract

The cellular uptake and localization of a Ru-octaarginine conjugate with and without an appended fluorescein are compared. The inherent luminescence of the Ru(II) dipyridophenazine complex allows observation of its uptake without the addition of a fluorophore. Ru-octaarginine-fluorescein stains the cytosol, nuclei, and nucleoli of HeLa cells under conditions where the Ru-octaarginine conjugate without fluorescein shows only punctate cytoplasmic labeling. At higher concentrations, however, Ru-octaarginine without the fluorescein tag does exhibit cytoplasmic, nuclear, and nucleolar staining. Attaching fluorescein to Ru-octaarginine lowers the threshold concentration required for diffuse cytoplasmic labeling and nuclear entry. Hence, the localization of the fluorophore-bound peptide cannot serve as a proxy for that of the free peptide.

Figure 1

Typical cellular distribution of Ru conjugates. HeLa cells were incubated with 5 μM Ru-D-R8 for 30 min (top), 5 μM Ru-D-R8-fluor for 30 min (middle) or 20 μM Ru-fluor for 41 h (bottom) at 37 °C in complete medium, then imaged by confocal microscopy. Structures of conjugates are shown at left. Note that Ru-D-R8 is isolated to the cytoplasm while Ru-D-R8-fluor stains the cytosol, nucleus and nucleoli. Ru-fluor shows only weak cytoplasmic staining. Scale bars are 10 μm. See Supplemental for wavelength comparisons.

Figure 2

Cellular distribution of Ru-D-R8 at higher concentration. HeLa cells were incubated with 20 μM Ru-D-R8 for 30 min at 37 °C in complete medium, rinsed with HBSS and imaged by confocal microscopy. The cells shown exclude the membrane-impermeable dead cell dye To-Pro-3. Some cells have only punctate staining of the cytoplasm (left) while others show additional staining of the nucleus and nucleoli as well as diffuse cytoplasmic staining (right). Scale bars are 10 μm.