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Cell fusion experiments reveal distinctly different association characteristics of cell-surface receptors

Péter Nagy1,2,3,*, László Mátyus1,*, Attila Jenei3,*, György Panyi1, Sándor Varga4, János Matkó1, János Szöllosi1, Rezso Gáspár1, Thomas M. Jovin3 and Sándor Damjanovich1,{ddagger}

1 Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
2 Cell-biophysical Workgroup of the Hungarian Academy of Sciences, Nagyerdei krt. 98, PO Box 39, H-4012 Debrecen, Hungary
3 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, D-37077 Göttingen, Germany
4 Central Research Service Laboratory, Medical and Health Science Center, University of Debrecen, H-4012 Debrecen, Hungary
* These authors contributed equally to the work



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  		Fig. 1. Intermixing of small- and large-scale protein clusters follows a different time course. Two samples of JY cells were labeled separately with fluoresceinated and rhodaminated anti-MHC-I Fabs (W6/32), respectively. The two separately labeled samples were subsequently fused. Temporal changes in FRET efficiency between fluoresceinated and rhodaminated W6/32 Fabs () and overlap of fluorescein and rhodamine clusters ({circ}) were measured. The filled bar on the right displays the FRET efficiency measured between fluoresceinated W6/32 and rhodaminated W6/32 on a double-labeled, non-fused JY cell sample, whereas the white bar shows the overlap of fluoresceinated W6/32 and rhodaminated W6/32 clusters on similarly labeled cells. Results are mean±s.e.m.

 


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  		Fig. 2. Scanning near-field optical and confocal microscopical images of PEG-fused cells. Cells were separately labeled with two different Fabs tagged with different fluorescent dyes. These samples were subsequently fused, and incubated at 37°C, except for D where incubation was on ice. Intermixing of the fluorescence in the two different channels was monitored using SNOM (A-F) and confocal (G-H) microscopy. The two images collected in the two fluorescence channels were superimposed on each other. The two signals are displayed in green and red, respectively. (A) Kit225 K6 cells, Cy3-IL-2R (green), Cy5-IL-2R (red), 0 minutes after fusion. (B) Kit225 K6 cells, Cy3-IL-2R (green), Cy5-IL-2R (red), 80 minutes after fusion. (C) JY cells, F-MHC-I (green), R-MHC-I (red), 80 minutes after fusion. (D) JY cells, F-MHC-I (green), R-MHC-I (red), 80 minutes after fusion, incubation on ice. (E) Kit225 K6 cells, Cy3-CD48 (green), Cy5-CD48 (red), 80 minutes after fusion. (F) Kit225 K6 cells, Cy3-TrfR (green), Cy5-TrfR (red), 80 minutes after fusion. (G) JY cells, F-MHC-I (green), R-MHC-I (red), 0 minutes after fusion, (H) JY cells, F-MHC-I (green), R-MHC-I (red), 80 minutes after fusion. Image size: A-F, 15x15 µm; G-H, 20x20 µm.

 


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  		Fig. 3. Intermixing of both small- and large-scale clusters of MHC-I and MHC-II proteins in the plasma membrane of JY cells points to their dynamic nature. Two samples of JY cells were separately labeled with antibodies tagged with different fluorescent labels. (For epitopes labeled by the antibodies see text below.) These samples were subsequently fused with each other using PEG. Cells were then incubated at 37°C in all the cases except where it is indicated. The FRET efficiency characterizing small-scale clustering of proteins was determined using the photobleacheing FRET approach 0 and 80 minutes after cell fusion (white and black columns, respectively, in A). Overlap of large-scale clusters was determined in a SNOM (B). When the two proteins labeled by the antibodies were different, overlapping cluster areas were normalized by dividing the double-colored area with the area of either of the protein clusters. Because the procedures yielded the same results within experimental error, only the normalization with the area of the first protein cluster is presented. As positive controls samples were labeled with antibodies against both proteins (left-hatched columns), and FRET efficiency and overlap percentage was determined similarly. Results are mean±s.e.m. Cell-surface receptor pairs were as follows: (A) MHC-I and MHC-I, (B) MHC-I and MHC-I, incubation on ice, (C) MHC-I and MHC-I in the presence of AlF3, (D) MHC-II and MHC-II, (E) MHC-I and MHC-II.

 


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  		Fig. 4. Raft and nonraft proteins home to their native cluster environment after PEG-induced fusion of Kit225 K6 human T cells. Cells were labeled and analyzed using the same procedures as those described in the legend to Fig. 3. Results are mean±s.e.m. A and B refer to data from FRET and SNOM experiments, respectively. Cell-surface receptor pairs were as follows: (A) CD48 and CD48, (B) TrfR and TrfR, (C) IL-2R {alpha} subunit and IL-2R {alpha} subunit, (D) IL-2 receptor {alpha} subunit and IL-2R receptor {alpha} subunit in the presence of AlF3, (E) MHC-I and MHC-I, (F) CD48 and TrfR.

 


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  		Fig. 5. Internalization of IL-2R is blocked by AlF3. Kit225 K6 cells were labeled with anti-Tac fab against the IL-2R. Cells were incubated in the absence (control sample, ) or in the presence ({circ}) of AlF3 at 37°C. Samples were taken every 20 minutes, and the ratio of intracellular fluorescence was determined with the acid-wash method as described in Materials and Methods. Results are mean±s.e.m.

 


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  		Fig. 6. Colloidal gold labeling of the {alpha} subunit of IL-2R reveals receptor intermixing after cell fusion. Kit225 K6 cells were labeled with anti-Tac antibodies against the IL-2R {alpha} subunit. Two samples of these cells were separately labeled with 10 and 30 nm colloidal gold beads, and fused with PEG. Samples were taken at 0 minutes (A) and 80 minutes (B) after cell fusion. (C) Nonfused Kit225 K6 cells labeled with both 10 and 30 nm colloidal gold beads. Bar, 200 nm.

 

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