Technical Reference #3049

3049.

HeLa Cell Entry by Guanidinium-Rich b-Peptides: Importance of Specific Cation–Cell Surface Interactions Terra B. Potocky; John Silvius; Anant K. Menon; and Samuel H. Gellman, University of Wisconsin Madison, ChemBioChem : a European Journal of Chemical Biology, 8(3049), (2007)
Link To Paper

Abstract:
Short cationic oligomers including arginine-rich peptides and analogous b-amino acid oligomers (“b-peptides”) can enter the cytoplasm and nucleus of a living cell from the extracellular medium. It seems increasingly clear that multiple entry pathways are possible depending upon the structure of the guanidiniumrich molecule the type of cell and other factors. We have previously shown that conformational stability and spatial clustering of guanidinium groups increase the HeLa cell entry efficiency of short helical b-peptides bearing six guanidinium groups results that suggest that these b-peptides could be useful tools for studying the entry process. Here we describe studies intended to identify the point in the entry process at which helix stability and spatial arrangement of guanidinium groups exert their effect. Our results suggest that key distinctions involve the mode of interaction between different guanidinium-rich b-peptides and the HeLa cell surface. Aspecifi c guanidinium display appears to be required for proper engagement of cell-surface heparan sulfate proteoglycans and concomitant induction of endocytic uptake.

Materials & Methods:
Confocal microscopy: HeLa cells grown to subconfluence on 90 mm plates were dissociated over a course of 15 min at 378C by treatment with trypsin/EDTA. Cells (105 per well) were plated onto 35 mm glass-bottomed culture dishes (MatTek) and cultured overnight in DMEM with FBS (10%) and pen/strep. The cells were then treated for the various experiments as described below. The cells were viewed by confocal microscopy by use of a BioRad MRC 1024 laser scanning confocal microscope with excitation/emission wavelengths set at 488/522 nm for fluorescein and 568/605 nm for propidium iodide. Peptide uptake was quantitated by cell counting. We distinguished among cells displaying only endosomal uptake (punctate fluorescence pattern) cells displaying cytoplasmic staining (diffuse fluorescence) and cells displaying nuclear localization; the last two categories overlapped almost perfectly. We generally saw little or no surface-localized fluorescence in treated cells. The number of cells showing nuclear uptake of carboxyfluorescein-labeled b-peptides was compared to the total number of cells as determined by transmission images. In each case 70–100 cells were evaluated. Each data point shown in the figures represents an average of at least four separate experiments. Propidium iodide (PI) staining was used to detect dead or dying cells; PI-stained cells were not included in the cell counting data (i.e. there were no PIstained cells among the 70–100 cells evaluated in a given experiment). In most cases we found that <10% of the cells treated with b-peptides were stained with PI indicating that the b-pep- ACHTUNGTRENUNGtides display little or no toxicity toward HeLa cells.

Microscopic Technique
Confocal microscopy

Cell Type(s)
HeLa cells