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First published online February 20, 2008
doi: 10.1242/10.1242/jcs.019513

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Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells

¹ Applied Optics group, Faculty of Science and Technology, MESA+ Research Institute for Nanotechnology, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands
² Cell Biology and Signaling Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, PO Box 39, 4032 Debrecen, Hungary
³ Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, PO Box 39, 4012 Debrecen, Hungary
⁴ Metabolism Branch/National Cancer Institute, National Institutes of Health, Bethesda MD 20892-1374, USA
⁵ ICFO-Institut de Ciències Fotòniques, 08860 Barcelona, Spain
⁶ ICREA-Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain
⁷ IBEC-Institut de Bioenginyeria de Catalunya and CIBER-BNN, Josep Samitier 1-5, Barcelona 08028, Spain

View larger version (137K): [in this window] [in a new window]   Fig. 1. (A) Bright-field image of fixed Kit 225 FT7.10 cells labeled with Cy5-conjugated antibodies against IL15R. Bar, 5 µm. (B) Confocal fluorescence image of the same sample area as in A. Bar, 5 µm. (C) NSOM image of the area highlighted in B, and magnified in D, demonstrating the increased spatial resolution and sensitivity of the technique as compared with that of the confocal method. The fluorescence signal on the images is color coded according to the detected polarization, red for 0° channel and green for 90° channel. The arrows in D point to some single-molecule spots. Individual molecules are identified by their unique dipole emission – that is, red and green color-coding. The yellow color of most fluorescent spots results from adding multiple molecules with random in-plane orientation (combination of red and green) in one spot and thus reflects the clustering of receptors on the cell membrane. Bars, 2 µm (C); 1 µm (D).

View larger version (135K): [in this window] [in a new window]   Fig. 2. Composite of four near-field fluorescence measurements performed on a Cy5-IL2R-labeled FT7.10 cell mapping the full distribution of the receptor on the membrane. The color coding of the image is the same as in Fig. 1. A compromise in image contrast accounts for the effect that the low-intensity spots in the middle of the cell are not clearly visible. The inset shows the central part of the cell with a different imaging contrast, revealing the presence of individual molecules (green and red spots corresponding to single dipole emission). Bar, 2 µm. The four independent NSOM images are shown in supplementary material Fig. S1.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Intensity distribution in terms of the fluorescence count rate and a normalized axis containing the number of Cy5 molecules for all images analyzed (see text for details) for (A) Cy5-IL2R and (B) Cy5-IL15R. The inset in A shows more accurately the low-intensity part of the distribution.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Domain area/size distributions for domains of IL2R (A) and IL15R (B) for all images analyzed. For convenience, both the occupied domain area (µm2) and the corresponding domain diameter are indicated on the horizontal axis of both distributions.

View larger version (14K): [in this window] [in a new window]   Fig. 5. (A) Correlation plots between domain area and the number of receptors contained in each domain for IL2R () and IL15R () for all images analyzed. The dotted lines are the lines of best fit to the correlation plots and suggest a constant packing density of both receptors on the cell membrane. (B) Distribution of the estimated intermolecular separations of IL2R and IL15R within each individual domain for all images analyzed.

View larger version (69K): [in this window] [in a new window]   Fig. 6. High-resolution dual-color excitation-detection NSOM of two different cells reflecting nanometer-scale spatial colocalization of IL2R (labeled with Alexa-Fluor-488-conjugated 7G7-B6 mAb) and IL15R (labeled with Cy5-conjugated 7A4 24 mAb) on the cell membrane. Here the pseudo-color scale refers to spectral contrast, with Alexa-Fluor-488 emission shown in green, whereas Cy5 emission is shown in red. The yellow color indicates colocalization of Alexa-Fluor-488 and the Cy5 signal. The degree of colocalization has been calculated using the Pearson's correlation coefficient over ten different cells imaged with NSOM (see Materials and Methods for details). Bars, 1 µm.