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James R. Johnson, Xudong Qiu, Edward M. Barnett, David Piwnica-Worms; Novel Caspase-Activated Cell-Penetrating Probes Reveal Endosomal Release During Apoptosis In a RGC-5 Cell Retinal Neurodegeneration Model. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5124.
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Caspase-activatable cell-penetrating peptide (CPP) probes show promise for characterizing retinal ganglion cell (RGC) apoptosis in vivo. These probes incorporate a CPP sequence (kkkrkv) and a fluorophore-quencher pair linked by a caspase-labile peptide sequence (DEVD). This scaffold displays efficient cell uptake and CPP probes are optically silent until cleavage by executioner caspases. Here, these probes were applied to RGC-5 cells to characterize the mechanisms underlying probe uptake, endosomal release and activation for correlation with probe activity observed with in vivo models of RGC neurodegeneration.
Our probes detected cell death in real-time facilitating the generation of time-lapse movies showing both the morphological changes of cells undergoing apoptosis and concurrent probe activation. To further characterize probe uptake and endosomal release, nonquenched probes Kcap488, KcapTR488 and Kcap647 (green, green/red and near infrared fluorescence, respectively) were synthesized and analyzed in RGC-5 cells using high-resolution fluorescence microscopy. For analysis of cell death, caspase-activatable probes KcapTR488, KcapQ488 and KcapQ647 were synthesized and tested in staurosporine and ionomycin cytotoxicity models.
RGC-5 cells internalize and retain CPP probes in endocytic compartments. High-resolution fluorescence microscopy revealed distinct probe (KcapQ488 and KcapQ647) activation in dying cells. Probe activation was confined to the cytosol and at no time after ionomycin or staurosporine treatment was endosomally associated signal observed. Thus, quenched probe escaped from endosomes into the cytosol during the course of apoptosis rather than being activated within endosomes prior to escaping. KcapTR488 facilitated multispectral imaging of endosomal probe release, subsequent cytosolic probe cleavage and differential subcellular trafficking of probe fragments with the progression of cell death. Independent confirmation of cell death was obtained via Annexin V co-staining as cells displaying probe activation were also bound by fluorescently-labeled Annexin V.
These caspase-activatable probes are apoptosis biosensors in RGC-5 cells. KcapTR488 facilitates monitoring of probe uptake, endosomal release and activation in RGC-5 neurodegeneration models. These studies demonstrate a mechanism by which caspase-activatable CPPs are internalized via endocytosis by living RGC-5 cells, released during apoptosis and activated during cell death as well as their utility for imaging apoptosis in RGCs.
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