Figure 1.
RPARPAR PAs internalize in PPC-1 cells in vitro to a higher extent than the peptide.
(A) Chemical structures of fluorescent peptides, peptide amphiphiles and control amphiphiles used in this study. (B) Quantification of cell-associated peptides, PAs and control amphiphile (concentration: 10 µM) after 1-hour incubation with PPC-1 cells revealed higher association for both types of RPARPAR PAs (2, 4) compared to peptide (1). Cell association was similar for RPARPAR PAs with carboxylated (2, 4) or amidated C-terminus (3, 5). Control amphiphile 6 and PA 7 showed lower association compared to RPARPAR PAs (2–5) Mean values and SEM are presented. (C) Confocal micrographs acquired under the same microscope settings confirmed elevated cellular uptake of PA 2 compared to peptide (1) and displayed a punctate intracellular fluorescence pattern. Nuclei stain (blue): Hoechst 33342; Scale bars: 40 µm.
Figure 2.
diC16-Rho-RPARPAR (2) co-localizes with CTb following 1-hour incubation in PPC-1 cells and is internalized in a dynamin-2-independent manner.
(A–D) PPC-1 cells incubated with PA 2 (10 µM) co-localized with CTb (A; yellow indicates co-localization) but not with mitochondria (B; Mitotracker) or lysosomes (C; Lysotracker). A small fraction of intracellular vesicles were positive for both PA 2 and transferrin (D). PPC-1 cells were transfected with EGFP-coupled dynamin-2 (E) or a dominant negative dynamin-2 mutant (G). 24 hours after transfection, cells were incubated for 1 hour with 10 µM PA 2. Absence of co-localization with dynamin-2 (E) and internalization in PPC-1 cells expressing the dominant negative dynamin-2 mutant (G) indicate that PA 2 enters cells in a dynamin-2-independent manner. Nuclei stain (blue): Hoechst 33342; Scale bars: 20 µm.
Figure 3.
Internalization of PAs requires cholesterol and is not inhibited by chlorpromazine or amiloride.
(A) PPC-1 cell association of PA 2 and PA 4 in presence of MβCD (cholesterol depletion agent) was reduced compared to controls Amiloride did not affect cell-association or internalization of PAs, whereas a low (10%) inhibition of cell-association was noted for PA 2 in presence of chlorpromazine. Average values and SEM are presented. (B) Qualitatively, the ratio of PAs localized on the plasma mebrane to the PAs found in intracellular vesicles was higher in MβCD treated-cells indicating that internalization was impaired. PA 2 associated with PPC-1 plasma membrane at 4°C but was not internalized after 1 hour. Scale bars: 20 µm.
Figure 4.
The non-peptidic part determines PA retention in PPC-1 cells.
(A) Pulse-chase experiments were performed with 10 µM PAs in PPC-1 cells with 1-hour pulse and different chase periods. Cell association of fluorescent PAs was determined and normalized (value of 1 corresponds to no chase). PPC-1-associated levels of PA 2 remained constant over 3 hours and decreased to half over 24 hours, whereas PA 4 levels decreased to 25% and 20% at 1 and 3 hours, respectively. PA 2 fluorescence values/well remained constant during a 24-hour chase (inset). Average values and standard deviations (n = 3) are presented. (B) Confocal micrographs of the two PAs at different chase points revealed similar intracellular patterns. Scale bars: 20 µM.
Figure 5.
RPARPAR PAs share the same initial internalization pathway and diverge at later time points.
Confocal micrographs at different time points of chase (pulse: 1 hour) revealed high extent of co-localization (yellow) between PA 8 (green) and PA 4 (red) when both PAs were chased for the same time (A,D). When PAs were chased for different time points, co-localization extent was decreased and was dependent on which PA was chased (B,C,E and see text for details). Nuclei stain (blue): Hoechst 33342. Scale Bars: 20 µM.
Figure 6.
RPARPAR PAs are recycled to the plasma membrane and trafficked to late endosomes.
Confocal micrographs of PPC-1 cells transfected with different EGFP-coupled Rab family members and incubated for 1 hour with 10 µM RPARPAR PAs. Both PAs (2 and 4) co-localized with Rab4-positive vesicles (A, B) and Rab7-positive vesicles (C, D). PA 4 additionally stained Rab11-positive vesicles (F). Arrows indicate examples of co-localization. Scale bars: 20 µM.
Figure 7.
diC16-Rho-RPARPAR (2) is eventually trafficked to lysosomes while DSPE-PEG2000-Rho-RPARPAR (4) is additionally found in CTb-positive vesicles after 24 hour incubation.
Confocal micrographs or RPARPAR PAs following a 1-hour pulse and 24-hour chase in PPC-1 cells treated either with CTb for 1 hour (A, B) or stained with lysotracker (C, D) showed differences in PA localization. PA 2 co-localized with lysosomes (C) but not CTb (A). PA 4 on the other hand, co-localized with both CTb (B) and lysosomes (D). Scale bars: 20 µm.
Figure 8.
Proposed model for internalization and trafficking of RPARPAR PAs.
Both PAs bind the plasma membrane and are taken up primarily via clathrin-independent pathways (solid line). Both PAs are recycled to the plasma membrane but diC16 PAs remains anchored to it (dashed line), whereas DSPE-PEG2000 PAs is washed away (dotted line). The diC16 tail is directed to lysosomes while DSPE-PEG2000 is trafficked both to lysosomes and CTb-containing organelles.