Caspase-Activated Cell-Penetrating Peptides Reveal Temporal Coupling Between Endosomal Release and Apoptosis in an RGC‑5 Cell Model

Abstract

Caspase-activatable cell-penetrating peptide (CPP) probes, designed for efficient cell uptake and specificity via cleavable intramolecular quenched-fluorophore strategies, show promise for identifying and imaging retinal ganglion cell apoptosis <i>in vivo.</i> However, initial cell uptake and trafficking events cannot be visualized because the probes are designed to be optically quenched in the intact state. To visualize subcellular activation events in real-time during apoptosis, a new series of matched quenched and nonquenched CPP probes were synthesized. In both native and staurosporine-differentiated RGC-5 cells, probe uptake was time- and concentration-dependent through clathrine-, caveolin-, and pinocytosis-mediated endocytic mechanisms. During apoptosis, KcapTR488, a novel dual fluorophore CPP probe, revealed by multispectral imaging a temporal coupling of endosomal release and effector caspase activation in RGC-5 cells. The novel CPPs described herein provide new tools to study spatial and temporal regulation of endosomal permeability during apoptosis