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, 8 (4), 389-401

Internalization and Trafficking of Cell Surface Proteoglycans and Proteoglycan-Binding Ligands

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Internalization and Trafficking of Cell Surface Proteoglycans and Proteoglycan-Binding Ligands

Christine K Payne et al. Traffic.

Abstract

Using multicolor live cell imaging in combination with biochemical assays, we have investigated an endocytic pathway mediated by cell surface proteoglycans, primary receptors for many cationic ligands. We have characterized this pathway for a variety of proteoglycan-binding ligands including cationic polymers, lipids and polypeptides. Following clathrin- and caveolin-independent, but flotillin- and dynamin-dependent internalization, proteoglycan-bound ligands associate with flotillin-1-positive vesicles and are efficiently trafficked to late endosomes. The route to late endosomes differs considerably from that following clathrin-mediated endocytosis. The proteoglycan-dependent pathway to late endosomes does not require microtubule-dependent transport or phosphatidyl-inositol-3-OH kinase-dependent sorting from early endosomes. The pathway taken by these ligands is identical to that taken by an antibody against heparan sulfate proteoglycans, suggesting that this mechanism may be used generally by cell surface proteoglycans and proteoglycan-binding ligands that lack secondary receptors.

Figures

Figure 1
Figure 1. Cellular entry of cationic polymers, lipids, and polypeptides is mediated by cell surface proteoglycans
A) Transfection of BS-C-1 cells by PEI and LF is inhibited by sodium chlorate, a drug that inhibits proteoglycan synthesis, in a concentration dependent manner. B) Transfection by PEI and LF is much less efficient in a proteoglycan-deficient cell line of CHO cells (PGD-CHO) than in wildtype CHO cells. Transfection efficiencies are normalized against untreated cells. Based on experiments done in triplicate, the error in the transfection measurement is 10%. C) Sodium chlorate (NaClO3, 80 mM) treatment blocks the binding of PA-QDs and anti-HSPG, but not that of transferrin (Tfn), to the surface of BS-C-1 cells. The diffuse red signal in the images is due to out of focus fluorescence on the thicker regions of the cells. Scale bars: 10 μm.
Figure 2
Figure 2. Polyplexes colocalize with a heparan sulfate antibody (anti-HSPG)
A) Polyplex (green) and anti-HSPG (red) after 20 minutes incubation with cells. Colocalization is shown in yellow. B) Tranferrin (green) and anti-HSPG (red) after 20 minutes incubation with cells. Scale bars: 10 μm. C) Percent colocalization of polyplexes and transferrin with anti-HSPG averaged from 5 to 30 minutes following binding.
Figure 3
Figure 3. Endocytosis of polyplexes, lipoplexes, PA-QDs, and anti-HSPG is independent of clathrin or caveolin
A) Microtubule-dependent motion (blue) of a vesicle containing polyplexes inside a cell. About 95% of vesicles containing polyplexes exhibit this type of movement. This rapid movement is inhibited when microtubules are disrupted with nocodazole (red). B) Amount of polyplexes, lipoplexes, PA-QDs, anti-HSPG, low-density lipoprotein (LDL), and cholera toxin B (CTB) that entered cells within 30 minutes for untreated cells (control) and cells treated with chlorpromazine or filipin. All results are normalized against untreated cells. C) Representative images of control cells transfected with non-specific siRNA, cells transfected with siRNA against clathrin heavy chain (CHC), and cells transfected with siRNA against caveolin-1 (Cav) 30 minutes following the addition of Alexa647-labeled transferrin (Tfn) or CTB. Transferrin or CTB that remained on the cell surface was removed with an acid wash. Scale bars: 10 μm. D) Fraction of polyplexes, anti-HSPG, transferrin (Tfn), and CTB that have entered cells within 30 minutes in control cells transfected with non-specific siRNA and clathrin or caveolin-1 knockdown cells.
Figure 4
Figure 4. Internalization of polyplexes requires dynamin-2
A) Fraction of transferrin and polyplexes that have entered cells within 30 minutes in control cells and Dynasore-treated cells. B) Fraction of transferrin and polyplexes that have entered cells within 30 minutes in control cells transfected with non-specific siRNA and in cells transfected with siRNA against dynamin-2.
Figure 5
Figure 5. Cationic ligands and anti-HSPG colocalize with flotillin-1 at early times post-endocytosis
A) PEI (red) and EGFP-flotillin-1 (green) at 10 minutes post-endocytosis. Colocalization is shown in yellow. B) Transferrin (red) and EGFP-flotillin-1 (green) at 10 minutes post-endocytosis. Scale bars: 10 μm. C) PEI, lipoplexes, PA-QDs, and anti-HSPG all show extensive colocalization with flotillin-1 vesicles within the first 20 minutes of entry. In comparison, transferrin and LDL, both clathrin-dependent ligands show less than 10% colocalization with flotillin-1. D) Fraction of transferrin and polyplexes that have entered cells within 30 minutes in control cells transfected with non-specific siRNA and in cells transfected with siRNA against flotillin-1.
Figure 6
Figure 6. Proteoglycans and proteoglycan-binding ligands are trafficked to late endosomes with high efficiency
A) Colocalization between EYFP-Rab5 (green) and EEA1 (red), an early endosomal marker. EEA1 was detected by immunofluorescence. Image adapted from Lakadamyali, et al. (43). B) Colocalization between EYFP-Rab9 (green) and DiD-labeled LDL (red). The image was taken 1 hour after LDL internalization, at which point LDL accumulates in late endosomes. C) A two-color image of polyplexes and EYFP-Rab5, an early endosomal marker, 20 minutes after the addition of polyplexes to cells. D) A two-color image of polyplexes and EYFP-Rab9, a late endosomal marker, 1 hour after the addition of polyplexes to cells. Colocalization is shown in yellow. Scale bars: 10 μm. E) Fraction of polyplexes colocalized with Rab5 (gray) and Rab9 (black) as a function of time. The solid and dashed lines indicate results from cells treated with nocodazole and untreated cells, respectively. . Error bars indicate standard error. Results were averaged over 5 cells. F) Fraction of LDL particles colocalized with Rab5 (gray) and Rab9 (black) as a function of time in untreated cells. Error bars indicate standard error. Results were averaged over 4 - 7 cells.
Figure 7
Figure 7. Proteoglycans and proteoglycan-binding ligands are trafficked to late endosomes independently of microtubule-dependent motion or PI(3)K-dependent sorting from early endosomes
A) Fraction of polyplexes, lipoplexes, PA-QDs, anti-HSPG, and LDL colocalized with Rab5 (gray) and Rab9 (black) at 1 hour after incubation with nocodazole-treated cells (solid columns). The majority of polyplexes, lipoplexes, PA-QDs, and anti-HSPG accumulate in endosomes that contain only Rab 9 but not Rab5. In contrast, the majority of LDL particles colocalize with both Rab5 and Rab9. The dashed columns show results for LDL in untreated control cells for comparison. Error bars indicate standard deviation. G) PtdIns3P-dependent sorting from early endosomes is not required for late endosomal entry of cationic ligands and HSPG. Fraction of polyplexes, lipoplexes, PA-QDs, and anti-HSPG in late endosomes that contain Rab9, but not Rab5, is similar in untreated cells and in cells treated with wortmannin, an inhibitor of PI(3)K. In comparison, colocalization of LDL with Rab9 is substantially reduced by wortmannin treatment. Measurements were done 1 hour post-endocytosis. Error bars indicate standard deviation.

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