Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy

Abstract

The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine.

Keywords: Autophagy; Endocytosis; Exocytosis; Nanomedicine; Protein nanocapsules..

Figure 1

Formation of nBSA. (A) Schematic illustration of nBSA. (B) Gel electrophoresis results of BSA and nBSA. (C) TEM image of nBSA. (D) Size distribution of nBSA determined with DLS. (E) Zeta potential distribution of nBSA. Scale bars: 50 nm.

Figure 2

The nBSA enters the cells through Arf-6 dependent endocytosis. (A, B) Confocal images of MCF-7 cells, which were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 hours. Arf-6 and EEA1 were detected with primary antibodies against Arf-6 and EEA1, respectively. (C, D, E) DsRed-Rab5, DsRed-Rab7, DsRed-Rab9 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (F) For lysosome detection, the MCF-7 cells were treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h and then co-treated with Lyso-Tracker Red probes for 30 min. Scale bars: 10 μm.

Figure 3

The nBSA enters the cells through the micropinocytosis-LEs-lysosome pathway. (A, B) DsRed-Rab34 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h; DsRed-Rab34 cells were co-transfected with EGFP-Rab7. (C-F) DsRed-Rab18 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h; EEA1 was then detected with a primary antibody against EEA1; DsRed-Rab18 cells were co-transfected with EGFP-Rab7; for lysosome detection, the MCF-7 cells were transfected with DsRed-Rab18 and then co-treated with Lyso-Tracker Red probes for 1 h. Scale bars: 10 μm.

Figure 4

nBSA through the recycling pathway. (A, B) DsRed-Rab11 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h; DsRed-Rab35 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (C, D) DsRed-Rab20 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h; DsRed-Rab25 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. Scale bars: 10 μm. (E) Schematic representation of the pathway through which the BSA-nanocapsules enter the cells, turnover in the cells and transport out of the cells.

Figure 5

nBSA through the exocytosis pathway. (A) DsRed-Rab8 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled nBSA for 20 h; DsRed-Rab10 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (B) DsRed-Rab22 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled nBSA for 20 h; DsRed-Rab31 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled BSA-nanocapsules for 20 h. (C) Schematic representation of how the cells use the Rab8, Rab10-positived GLUT4 transport vesicles pathway to transport protein nanocapsules out of the cells.

Figure 6

nBSA induces autophagy and are sequestered by the autophagosomes. (A) Representative images and quantification of MCF-7 cells with EGFP-LC3 vesicles (autophagosomes). EGFP-LC3 transfected cells were treated with 1 mg/mL non-labeled nBSA for 20 h. Scale bars: 10 μm. Data are provided as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.0001 compared to controls. (B) LC3I/II protein levels were analyzed by western blotting in the MCF-7 cells treated in (A). (C) DsRed-LC3 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled nBSA for 20 h; MCF-7 cells were treated with 1 mg/mL FITC-labeled nBSA for 20 h and then P62 was detected with a primary antibody against P62. (D) DsRed-LC3 transfected MCF-7 cells and then P62 was detected with a primary antibody against P62. (E) EGFP-LC3 transfected MCF-7 cells were co-treated with Lyso-Tracker Red probes for 1 h. The autophagosomes fuse with lysosomes (arrows). The above images are enlarged from the white outline in the images below. Scale bars: 10 μm.

Figure 7

Crosstalk between endocytosis, exocytosis and autophagy. (A-H) EGFP-LC3 cells were co-transfected with DsRed-Rab23, DsRed-Rab34, DsRed-Rab7, DsRed-Rab18, DsRed-Rab11, DsRed-Rab35, DsRed-Rab8 and DsRed-Rab10, respectively. Scale bars: 10 μm.

Figure 8

Inhibiting autophagy and exocytosis enhances the effect of protein nBSA. (A, B) DsRed-LC3 transfected MCF-7 cells were treated with 1 mg/mL FITC-labeled nBSA for 20 h; DsRed-LC3 cells were co-transfected with Flag-vBcl-2 and then treated with 1 mg/mL FITC-labeled nBSA for 20 h. (C, D) Representative images of MCF-7 cells with DsRed-LC3 vesicles. The DsRed-LC3 transfected MCF-7 cells were co-treated 10 μM 3-MA and 30 μM CQ, respectively. (E, F) DsRed-Rab7 transfected MCF-7 cells were then treated with 1 mg/mL FITC-labeled nBSA for 20 h; The DsRed-Rab7 transfected MCF-7 cells were co-treated with 30 μM CQ. Scale bars: 10 μm.

Figure 9

Schematic representation of the degradation pathway of protein nanocapsules in cell. Rab34 positive micropinocytosis, Rab7 and Rab9 positive late endosome, Rab18 positive liposome, Rab11 and Rab35 positive slow recycling endosome, Rab8 and Rab10 positive GLUT4 vesicles co-localize with autophagosomes.