A Single GUV Method for Revealing the Action of Cell-Penetrating Peptides in Biomembranes.

ABSTRACT

The mechanism of entry of cell-penetrating peptides (CPPs) into the cytosol of various cells has been studied by examining the interaction of CPPs with lipid bilayers and their entry into lipid vesicle lumens using various methods. Here we describe a single giant unilamellar vesicle (GUV) method to study CPPs. In this new method, we use GUVs containing small GUVs in the mother GUV lumen or GUVs containing large unilamellar vesicles (LUVs) in the GUV lumen and investigate the interaction of fluorescent probe-labeled CPPs with single GUVs in real time using confocal laser scanning microscopy. This method can detect CPPs in the GUV lumen with high sensitivity, allowing immediate measurement of the time course of entry of CPPs into the vesicle lumen. This method allows simultaneous measurement of the entry of CPPs and of CPP-induced pore formation, allowing the relationship between the two events to be determined. One can also simultaneously measure the entry of CPPs and the CPP concentration in the GUV membrane. The rate of entry of CPPs into a single GUV lumen can be estimated by obtaining the fraction of GUVs into which CPPs entered before a specific time t without pore formation among all examined GUVs (i.e., the fraction of entry) and the lumen intensity due to LUVs with bound CPPs. This method is therefore useful for elucidating the mechanism of entry of CPPs into lipid vesicles.