Insight into cell-entry mechanisms of CPPs by electron microscopy.
Despite the quickly widening application of cell-penetrating peptides (CPP) for the cellular delivery of various macromolecules, the cell entry mechanisms of these peptides have remained elusive so far. The basic features of the translocation of CPPs into cells have been mapped by fluorescence microscopy and activity-based assays revealing that endocytotic mechanisms are mainly responsible for the uptake at physiological temperature. However, the high concentration of CPP or the lowering of the incubation temperature below 10°C (re)activates a nonvesicular cell entry mode. The fluorescence microscopy can hardly provide detailed information about the interaction of CPP molecules with the extracellular structures, the induced changes in the morphology of the plasma membrane, etc. Therefore, application of electron microscopy could help to shed light on the nature of nonvesicular uptake mechanism. Transmission electron microscopy (TEM) has been a valuable tool for the morphological characterization of biological material at high resolution. It can provide useful information at the ultrastructural level about the interaction and arrangement of CPPs on the cell surface, the entrapment in cellular organelles and the translocation to the cytoplasm. In this chapter, we present a method for the tagging of CPPs covalently with a 1.4 nm gold cluster and provide a flat-embedding protocol for the mapping of Nanogold™-labeled CPPs in cultured cells by TEM. This method enables to retain the cell monolayers in their in situ orientation. The Nanogold™ tag is putatively not interfering with the uptake of CPPs and enables the production of specimens with excellent morphology and good contrast.
Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
Kärt
K
Padari
Annely
A
Lorents
Eija
E
Jokitalo
Margus
M
Pooga
D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis; D006046:Gold / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D012834:Silver / Q000737:chemistry; D013194:Staining and Labeling
D057846:Cell-Penetrating Peptides; D012834:Silver; D006046:Gold