Limits...
Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

He Q, Sun X, Chu C, Jiang Q, Zhu H, He Y, Yue T, Wang R, Lei P, Shen G - PLoS ONE (2015)

Bottom Line: Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis.The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles.This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

ABSTRACT
The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

No MeSH data available.


Related in: MedlinePlus

Dynamics of hTfR-EGFP mediated endocytosis in living cells.CHO-hTfR cells expressing hTfR-EGFP were grown in glass chambers overnight. (A) Cells were cultured with anti-hTfR mAb for 1h at 4°C, and then the chamber was mounted onto the microscope stage. 2 min after the supplement of secondary antibody conjugated with PE to the chamber, image acquisition of PE and EGFP fluorescences was performed. Images were acquired continuously at 2-min interval for 40min on a microscope stage. Presented were the selected images taken during this period. Images were indicated by time points. PE and EGFP segregation emerged from 18 min. (B and C) Anti-TfR mAb (AMCA secondary labeling) binding cells were incubated for another 5min or 30min at 37°C to allow internalization. PE conjugated anti- mouse LAMP-1/ EEA-1 antibody was used to label intracellular lysosome and endosome to show the colocalization with mAb and hTfR-EGFP chimera at different time point. EEA-1 for 5min and LAMP-1 for 30min.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4372551&req=5

pone.0122452.g005: Dynamics of hTfR-EGFP mediated endocytosis in living cells.CHO-hTfR cells expressing hTfR-EGFP were grown in glass chambers overnight. (A) Cells were cultured with anti-hTfR mAb for 1h at 4°C, and then the chamber was mounted onto the microscope stage. 2 min after the supplement of secondary antibody conjugated with PE to the chamber, image acquisition of PE and EGFP fluorescences was performed. Images were acquired continuously at 2-min interval for 40min on a microscope stage. Presented were the selected images taken during this period. Images were indicated by time points. PE and EGFP segregation emerged from 18 min. (B and C) Anti-TfR mAb (AMCA secondary labeling) binding cells were incubated for another 5min or 30min at 37°C to allow internalization. PE conjugated anti- mouse LAMP-1/ EEA-1 antibody was used to label intracellular lysosome and endosome to show the colocalization with mAb and hTfR-EGFP chimera at different time point. EEA-1 for 5min and LAMP-1 for 30min.

Mentions: The characterization of stable expression of hTfR-EGFP described above suggests that this chimera is indeed an appropriate tool to study hTfR trafficking using optical microscopy of live cells. In following experiments the mAb-induced endocytosis of hTfR-EGFP was visualized in CHO-hTfR cells. An important question that could now be addressed was to compare the post-endocytic localization of the monoclonal antibody, and the hTfR. Cells were allowed to internalize anti-hTfR mAb labeled using PE-conjugated secondary Ab at 37°C, and the time course of cellular distribution of EGFP and PE red fluorescence was monitored in living cells using Olympus imaging system. As shown in Fig 5A, the binding of mAb and its diffuse staining on the cell surface were clearly seen after 2 min of Ab addition. The fluorescent images were acquired following additional 38 min of continuous endocytosis at 37°C. After 6–8 min of incubation, the evenly distributed fluorescence first became ‘grainy’, thereafter, a large part of the grains started to cluster. Green fluorescence was seen to colocalize with internalized red fluorescence and the resulted orange/yellow fluorescence clustered in the same submembrane vesicular structures. The endosomal compartments were rapidly moving and often migrating toward a perinuclear area and had a complex morphology. At the late stages of intracellular trafficking, green and some red fluorescence remained co-localized in the large perinuclear endosomes. However, a small portion of PE fluorescence was associated with the peripheral vesicular compartments that did not contain EGFP (emerged from 18min).


Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

He Q, Sun X, Chu C, Jiang Q, Zhu H, He Y, Yue T, Wang R, Lei P, Shen G - PLoS ONE (2015)

Dynamics of hTfR-EGFP mediated endocytosis in living cells.CHO-hTfR cells expressing hTfR-EGFP were grown in glass chambers overnight. (A) Cells were cultured with anti-hTfR mAb for 1h at 4°C, and then the chamber was mounted onto the microscope stage. 2 min after the supplement of secondary antibody conjugated with PE to the chamber, image acquisition of PE and EGFP fluorescences was performed. Images were acquired continuously at 2-min interval for 40min on a microscope stage. Presented were the selected images taken during this period. Images were indicated by time points. PE and EGFP segregation emerged from 18 min. (B and C) Anti-TfR mAb (AMCA secondary labeling) binding cells were incubated for another 5min or 30min at 37°C to allow internalization. PE conjugated anti- mouse LAMP-1/ EEA-1 antibody was used to label intracellular lysosome and endosome to show the colocalization with mAb and hTfR-EGFP chimera at different time point. EEA-1 for 5min and LAMP-1 for 30min.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372551&req=5

pone.0122452.g005: Dynamics of hTfR-EGFP mediated endocytosis in living cells.CHO-hTfR cells expressing hTfR-EGFP were grown in glass chambers overnight. (A) Cells were cultured with anti-hTfR mAb for 1h at 4°C, and then the chamber was mounted onto the microscope stage. 2 min after the supplement of secondary antibody conjugated with PE to the chamber, image acquisition of PE and EGFP fluorescences was performed. Images were acquired continuously at 2-min interval for 40min on a microscope stage. Presented were the selected images taken during this period. Images were indicated by time points. PE and EGFP segregation emerged from 18 min. (B and C) Anti-TfR mAb (AMCA secondary labeling) binding cells were incubated for another 5min or 30min at 37°C to allow internalization. PE conjugated anti- mouse LAMP-1/ EEA-1 antibody was used to label intracellular lysosome and endosome to show the colocalization with mAb and hTfR-EGFP chimera at different time point. EEA-1 for 5min and LAMP-1 for 30min.
Mentions: The characterization of stable expression of hTfR-EGFP described above suggests that this chimera is indeed an appropriate tool to study hTfR trafficking using optical microscopy of live cells. In following experiments the mAb-induced endocytosis of hTfR-EGFP was visualized in CHO-hTfR cells. An important question that could now be addressed was to compare the post-endocytic localization of the monoclonal antibody, and the hTfR. Cells were allowed to internalize anti-hTfR mAb labeled using PE-conjugated secondary Ab at 37°C, and the time course of cellular distribution of EGFP and PE red fluorescence was monitored in living cells using Olympus imaging system. As shown in Fig 5A, the binding of mAb and its diffuse staining on the cell surface were clearly seen after 2 min of Ab addition. The fluorescent images were acquired following additional 38 min of continuous endocytosis at 37°C. After 6–8 min of incubation, the evenly distributed fluorescence first became ‘grainy’, thereafter, a large part of the grains started to cluster. Green fluorescence was seen to colocalize with internalized red fluorescence and the resulted orange/yellow fluorescence clustered in the same submembrane vesicular structures. The endosomal compartments were rapidly moving and often migrating toward a perinuclear area and had a complex morphology. At the late stages of intracellular trafficking, green and some red fluorescence remained co-localized in the large perinuclear endosomes. However, a small portion of PE fluorescence was associated with the peripheral vesicular compartments that did not contain EGFP (emerged from 18min).

Bottom Line: Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis.The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles.This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.

ABSTRACT
The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

No MeSH data available.


Related in: MedlinePlus