Abstract:Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequ… Show more
“…A similar situation can occur inside cells, provided that a concentration threshold is reached in confined regions of the cell leading to fluorescence self-quenching as we demonstrated herein, which deleterious effect on the fluorescent signal can be further amplified in acidic compartments such as endosomes. This kind of quenching was also recently reported for confocal laser scanning microscopy experiments33. Quenching might also contribute to the small difference (1.25 to 2 folds) observed between the total cell-associated and the internalized fluorescence signals measured by flow cytometry for penetratin, contrasting with the ratio obtained by fluorometry and mass spectrometry, close to 100 folds for this peptide.…”
The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.
“…A similar situation can occur inside cells, provided that a concentration threshold is reached in confined regions of the cell leading to fluorescence self-quenching as we demonstrated herein, which deleterious effect on the fluorescent signal can be further amplified in acidic compartments such as endosomes. This kind of quenching was also recently reported for confocal laser scanning microscopy experiments33. Quenching might also contribute to the small difference (1.25 to 2 folds) observed between the total cell-associated and the internalized fluorescence signals measured by flow cytometry for penetratin, contrasting with the ratio obtained by fluorometry and mass spectrometry, close to 100 folds for this peptide.…”
The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.
“…For the TMR and AF555 conjugates, an increase in the fluorescence is observed upon irradiation (Figs 4 and 5 ). This phenomenon is most likely caused by higher self-quenching in the endosomes, where the fluorophore concentration is much higher than in the cytoplasm after release 33 . Figure 4 Relocalization of PNA conjugates from endosomes to cytosol upon irradiation.…”
Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginine peptide nucleic acid (PNA) conjugates were investigated in terms of PCI assisted cellular activity. It is found that tetramethylrhodamine and Alexa Fluor 555 conjugated octaarginine PNA upon irradiation exhibit more than ten-fold increase in antisense activity in the HeLa pLuc705 luciferase splice correction assay. An analogous fluorescein conjugate did not show any significant enhancement due to photobleaching, and neither did an Alexa Fluor 488 conjugate. Using fluorescence microscopy a correlation between endosomal escape and antisense activity was demonstrated, and in parallel a correlation to localized formation of ROS assigned primarily to singlet oxygen was also observed. The results show that tetramethylrhodamine (and to lesser extent Alexa Fluor 555) conjugated octaarginine PNAs are as effectively delivered to the cytosol compartment by PCI as by chloroquine assisted delivery and also indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore.
“…In cells, they took advantage of the IR modality of the probe to quantify the internalization of the conjugates. They could also show that apparent discrepancies with fluorescence quantification were due to a variable fluorescence enhancement of the probes in membrane environment, which hampered rigorous quantification [13,[103][104][105][106]. In skin biopsies, the IR modality also enabled them to quantify the penetration of the peptide conjugate into skin, while the fluorescence modality could be used to compare its localization with those of nuclei, using common fluorescence stain like DAPI.…”
Section: N-terminal Labelling Of Peptidementioning
Bio-imaging, by enabling the visualization of biomolecules of interest, has proved to be highly informative in the study of biological processes. Although fluorescence microscopy is probably one of the most used techniques, alternative methods of imaging, providing complementary information, are emerging. In this context, metal complexes represent valuable platforms for multimodal imaging, since they may combine interesting spectroscopic features and biologically relevant functionalization on a single molecular core. In particular, d6 low-spin rhenium tri-carbonyl complexes display unique luminescence and vibrational properties, and can be readily functionalized. Here we review their applications and potential as probes or drugs relying on their photophysical properties, before focusing on their use as multimodal probes for the labelling and imaging of peptides and proteins.
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