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Part Number Referenced: P35GC-0-10-C
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TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS |
MICROSCOPY |
SPECIES |
MORPHOLOGY |
CELL LINE |
639
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Fluorescent
dyes alter intracellular targeting and function of cell-penetrating tetrapeptides |
Hazel
H. Szeto, Peter W. Schiller, Kesheng Zhao, and Guoxiong Luo |
membrane
transduction, mitochondria, permeability transition, cytoplasm, confocal microscopy |
Caco-2 cells were plated on 35
mm glass bottom dishes (MatTek, Ashland, MA) for 2 days.
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confocal
laser scanning microscopy |
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epithelial |
Caco-2 |
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Fluorescent labels are commonly used to investigate the mechanisms of cellular
uptake and intracellular distribution of cell-penetrating peptides. However, labels such as
fluorescein and rhodamine are relatively large and very lipophilic and may significantly alter
physicochemical properties of small peptides. To minimize the impact of the fluorescent probe
on a tetrapeptide, we substituted one of the amino acids (Lys4) in a tetrapeptide ([Dmt1]DALDA,
Dmt-D-Arg-Phe-Lys-NH2 where Dmt = 2’,6’-dimethyltyrosine) with two different fluorescent
amino acids (ß-dansyl-L-a,ß-diaminopropionic acid (dnsDap4) or ß-anthraniloyl-L-a,ß-diaminopropionic
acid(atnDap4)). Initial studies with confocal laser scanning microscopy (CLSM) showed very different
localization patterns for the two fluorescent analogs, with [Dmt1,atnDap4]DALDA showing mitochondrial
localization and [Dmt1,dnsDap4]DALDA showing diffuse cytoplasmic localization. Studies with
isolated mouse liver mitochondria suggested that [Dmt1,dnsDap4]DALDA targeted the mitochondrial
matrix resulting in mitochondrial depolarization, opening of the permeability transition pore,
mitochondrial swelling, and rapid release of the peptide into the cytoplasm. In contrast, [Dmt1,atnDap4]DALDA
was retained in the inner mitochondrial membrane and did not induce mitochondrial swelling.
Furthermore, [Dmt1,atnDap4]DALDA protected mitochondria against Ca2+-induced swelling. Importantly,
the unlabeled parent peptide [Dmt1]DALDA behaved like [Dmt1,atnDap4]DALDA and was mitoprotective.
These findings suggest that experimental results obtained with fluorescent labels must be interpreted
with caution, and the use of multiple fluorophores, together with confirmation using the original
or radiolabeled molecule, is recommended. |
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