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First published online 10 February 2009
doi: 10.1242/jcs.031427

Journal of Cell Science 122, 600-610 (2009)
Published by The Company of Biologists 2009
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Physical transfer of membrane and cytoplasmic components as a general mechanism of cell-cell communication

Xinle Niu1, Kshitiz Gupta1, Joy T. Yang2, Michael J. Shamblott3 and Andre Levchenko1,*

1 Department of Biomedical Engineering, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA
2 Department of Cell Biology, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA
3 Department of Gynecology and Obstetrics, Johns Hopkins University, School of Medicine, Baltimore, MD 21205, USA


Figure 1
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  		Fig. 1. Physical transfer of membrane proteins in CHO cells. (A-C) Untransfected CHO cells (solid black lines) were co-cultured respectively with labeled cells (dashed pink lines): CHO-CD36+ cells (A), CHO-ICAM1 cells (B) and CHO-{alpha}4-GFP cells (C). Cell populations were separated by a 0.45 µm filter (dotted black lines), or were grown in co-culture (green lines). (D) Time course of CD36 transfer from CHO-CD36 cells to untransfected CHO cells before co-culture (blue), and after 1 day (green), 2 days (red), 3 days (cyan), 4 days (purple) and 5 days (light green). (E) Average fluorescence intensity per unit cell determined by epifluorescence in co-culture of CHO-CD36+ cells and CHO-{alpha}4-GFP cells. Each bar represents the average epifluorescence intensity of at least 50 cells.

 

Figure 2
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  		Fig. 2. Bidirectional transfer of membrane proteins between CHO-CD36+, CHO-ICAM1 and CHO-{alpha}4-GFP cells co-incubated a pair-wise fashion. (A) CD36 and {alpha}4-GFP transfer between CHO-CD36+ cells and CHO-{alpha}4-GFP cells. (B) ICAM-1 and {alpha}4-GFP transfer between CHO-ICAM1 cells and CHO-{alpha}4-GFP cells. (C) CD36 and ICAM-1 transfer between CHO-CD36+ cells and CHO-ICAM1 cells.

 

Figure 3
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  		Fig. 3. Properties of membrane protein transfer. Donor cells (CHO-CD36+ and CHO-ICAM-1) are represented by dashed pink lines, and acceptor CHO cells represented by thin black lines. (A) CHO-CD36+ cells co-cultured with CHO cells at the seeding ratio of 1:3.6 (thick green line) or 1.2:1 (thick blue line). (B) CHO-ICAM1 cells co-cultured with CHO cells at the ratio of 1:1 (thick blue line) or 1:2 (thick green line). (C) CHO-CD36+ cells and CHO cells co-cultured in a 75 cm2 flask at the ratio of 1:1 with initial cell numbers of 1x106 (thick green line) and 2x106 (thick blue line). (D) Transfer of membrane protein is transient. 5 days after separation from CHO-CD36 cells (thick blue line) by FACS, CHO-CD36+ cells (dashed pink line) and CHO-CD36– cells (thin black line) were co-cultured again (thick green line) Black dotted line, untransfected CHO cells. (E) CHO-ICAM1 cells and CHO cells co-cultured in the presence of linoleic acid (25 µM) (thick blue line) and without linoleic acid (dotted pink line). (F) CHO-CD36 and CHO cells co-cultured without (thick red line) and in the presence of methyl-β-cyclodextrin (1 mM) (thick green line) for 2 days.

 

Figure 4
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  		Fig. 4. Generality of membrane protein transfer. Human cells expressing high levels of certain membrane proteins [P-gp on BE(2)C/CHC(0.2) cells, ErbB2 on SKOV3 cells and EGFR A431 cells] and stably transfected CHO cells CHO-CD36+, CHO-ICAM1 and CHO-{alpha}4-GFP cells were co-cultured with each other or with other recipient cells (PC12 cells, NIH 3T3 cells, untransfected CHO cells, HUVECs, iHUVECs, LVECs and GMSCs). After each co-culture, cells were labeled with fluorescence-conjugated monoclonal antibodies for specific membrane proteins, followed by flow cytometry measurement. This diagram summarizes the results of the co-culture experiments in this study (see supplementary material Fig. S1 and Figs 1, 3 and 5 for the corresponding data). A solid line represents transfer of a specific protein in a co-culture experiment with its arrow pointing to the recipient cells, whereas a dashed line represents no transfer detected in a co-culture experiment. All primary antibodies were raised from mouse and only recognized the extracellular domain of the transmembrane proteins.

 

Figure 5
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  		Fig. 5. Intercellular transfer of membrane lipids. (A) PKH67-labeled CHO-CD36+ cells (dashed pink line) were co-cultured with unlabeled CHO cells (solid black line) in a monolayer (thick green line) or separated by a filter with 0.45 µm pores (thick black line). (B) PKH67-labeled SKOV3 cells were co-cultured with unlabeled SKOV3 cells (white arrows). (C) PKH67-labeled CHO-CD36+ cells were co-cultured with CHO cells. (D) PKH67-labeled CHO-CD36+ cells were co-cultured with A431 cells. (E) PKH67-labeled A431 cells were co-cultured with CHO-CD36+ cells. CD36 and EGFR levels in C-E were determined by flow cytometry.

 

Figure 6
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  		Fig. 6. Analysis of intercellular transfer of cytoplasmic components by flow cytometry. (A-L) Unlabeled CHO cells (solid black line) and CHO-CD36+ cells (dashed pink line) labeled with calcein-AM, Fluorescein-conjugated dextrans of different molecular sizes and quantum dots, were co-cultured (thick green line), separated with a 0.45 µm filter (dotted blue line), and with higher ratios of labeled to unlabeled cells (thick blue lines). (M-N) Cytoplasmic macromolecules and membrane proteins were co-transferred onto recipient cells (M) or mutually transferred between co-cultured cells (N).

 

Figure 7
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  		Fig. 7. Stem cells can accept large cytoplasmic macromolecules from somatic cells. (A-D) Primary HUVECs labeled with 10 kDa and 2000 kDa Fluorescein-conjugated dextrans (dashed line) were co-cultured with NIH 3T3 cells or uncommitted LVECs (thin black lines), and co-cultures (gray lines) studied by flow cytometry. (E,F) iHUVECs transfected stably with GFP were co-cultured with LVECs (E) and CHO-CD36+ cells (F).

 

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