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First published online February 20, 2008
doi: 10.1242/10.1242/jcs.019513
Research Article |
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
* Authors for correspondence (e-mail: jenei{at}jaguar.dote.hu; mgarcia{at}pcb.ub.es)
Accepted 28 November 2007
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-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2R and IL15R at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2R and IL15R at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2R molecules reside outside the clustered domains, whereas 
30% of IL15R molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2R and IL15R domains remained constant, suggesting a `building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two -chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.
Key words: Near-field scanning optical microscopy (NSOM), Interleukin receptors IL2R, IL15R, Single-molecule detection, nanometer-scale membrane organization
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). Their respective receptors comprise three distinct components: the -chains are cytokine specific, whereas the β- and 
c-subunits are utilized by both IL2 and IL15. In addition, the so-called common c-chain is a component of a series of other cytokine receptors, these being members of the c cytokine receptor family (IL4, IL7, IL9, IL21) (Nakamura et al., 1994; Nelson and Willerford, 1998; Fehniger and Caligiuri, 2001; Tagaya et al., 1996). As a result of combining the various subunits, several forms of receptor complexes with different affinities to the interleukins might exist at the cell surface. The trimeric receptor complexes (βc) of both IL2 and IL15 have similar high affinities for their ligands (Kd 10–11 M). The βc heterodimer has an intermediate affinity for both cytokines (Kd 10–9 M). In contrast to IL2 receptor (IL2RA, hereafter referred to as IL2R), which on its own binds to IL2 with very low affinity (Kd 10–8 M), IL15 receptor (IL15RA, hereafter referred to as IL15R) is capable of binding to IL15 with a high affinity matching that of the trimeric IL15R (Lin et al., 1995).
Heterodimerization of the intracellular domains of the β- and c-chains is crucial for one set of signaling events shared by both cytokines (Nakamura et al., 1994; Fehniger et al., 2001). In this case, IL2 and IL15 activate similar signaling pathways involving Janus kinase (JAK1/JAK3)-assisted tyrosine phosphorylation of downstream signaling molecules (e.g. signal transducer and activator of transcription molecules STAT3 and STAT5) (Lin et al., 1995). As IL2R does not possess signaling capacity, IL2-induced signals are transmitted solely by the IL2Rβc heterotrimeric or the βc heterodimeric forms of the IL2R complexes and utilize the Jak-STAT-related pathways. By contrast, it appears that IL15 can stimulate additional signaling pathways employing IL15R but not requiring the expression of the βc heterodimer (Bulanova et al., 1995).
As a consequence of utilising shared receptor subunits and common signaling elements, IL2 and IL15 induce similar cellular responses in many cases (Waldmann et al., 2001). Among others, they both stimulate the proliferation of several T cell subsets and facilitate the induction of cytolytic effector T cells (Grabstein et al., 1994; Waldmann and Tagaya, 1999). However, they can also exhibit quite distinct and contrasting contributions to T-cell-mediated immunity. Through its central role in activation-induced cell death (AICD), IL2 is involved in peripheral tolerance to self-reactive T cells. IL15, by contrast, inhibits IL2-mediated AICD (Marks-Konczalik et al., 2000) and stimulates the persistence of CD8+ memory T cells (Ku et al., 2000). Many explanations have been proposed to resolve this apparent contradiction. It has been shown previously that, in the early phase of the immune response, IL15R expressed on antigen-presenting cells (e.g. monocytes, dendritic cells) can present the tightly associated IL15 in trans to a cytokine-responsive second cell (e.g. natural killer or memory T cell) expressing only the β and c chains (Dubois et al., 2002). By contrast, IL2 mainly acts as a soluble factor binding to receptor complexes on a single T cell. The existence of the aforesaid βc-independent signaling routes by IL15R alone is another possible explanation for the diverse outcomes of biological responses invoked by IL15 and IL2 (Bulanova et al., 2001).
On cells expressing all the elements of the IL2R-IL15R system, the diversity of downstream signaling might originate from the different molecular interactions of the two chains. However, our recent confocal laser scanning microscopy (CLSM) and fluorescence resonance energy transfer (FRET) experiments revealed the physical proximity of IL2R and IL15R as well as their association with the β- and c-subunits and with major histocompatibility complex (MHC) glycoproteins in lipid rafts of human T lymphoma/leukemia cells, indicating a considerable similarity between the direct molecular environment of the two -chains (Vámosi et al., 2004). However, it can be assumed that cytokine-specific modulation of subunit assembly within lipid rafts, and, as a result, the alteration of receptor affinity and/or conformation of the recruited signaling elements, might explain the distinct actions of IL2 and IL15. Indeed, based on our FRET data, a heterotetrameric model of the IL2-IL15R complex could be generated, where binding of IL2 or IL15 causes the formation of the appropriate high-affinity receptor trimer (βc), while the `unused' -chain moves away from the site of cytokine-receptor interaction (Vámosi et al., 2004). A distinct involvement of FK506-binding proteins 12 and 12.6 in IL2- versus IL15-mediated T cell responses has also been reported (Dubois et al., 2003).
Most of the proposed mechanisms that might underlie the divergence in IL2 and IL15 signaling are based on the unique -chains of the two cytokines. Therefore, we decided to investigate the organization of IL2R and IL15R on a human CD4+ leukemia T cell line, Kit 225 FT7.10, at high spatial resolution using near-field scanning optical microscopy (NSOM). NSOM is a technique based on lensless optical imaging that provides simultaneous optical and topographic lateral resolution beyond the diffraction limit of light (de Lange et al., 2001; Ianoul et al., 2005). The technique is based on local excitation of the sample using a sub-wavelength light source that is then raster-scanned over the surface. The lateral resolution is limited to the dimension of the aperture (typically 70 nm), whereas the axial resolution is defined by the diffraction of the evanescent field emanating from the aperture (<50 nm), resulting in an overall reduction of the illumination volume by a factor of >100 below the diffraction limit. As such, the technique is particularly suitable for investigating the distribution of membrane components on the cell surface with nanometer precision and single-molecule detection sensitivity (de Lange et al., 2001; Koopman et al., 2004).
As an extension of our previous CLSM experiments reporting on the lateral distribution and colocalization of IL2R and IL15R at the submicron scale (Vámosi et al., 2004; Vereb et al., 2000), we now provide detailed analysis at a finer spatial scale, confirming clustering of IL2R and IL15R on the cell membrane. Moreover, the single-molecule-detection sensitivity of our NSOM technique also provides insight into the inner structure of these domains and has allowed us to derive quantitative analysis of the packing densities and intermolecular distances between their components. Furthermore, information on the fraction of proteins residing inside or outside the domain regions has been obtained. Finally, simultaneous excitation of both IL2R and IL15R with the same NSOM probe showed co-clustering at the nanometer scale, supporting previous FRET and CLSM observations.
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and IL2R in the plasma membrane of FT7.10 cells and IL2R was investigated independently, that is in different cells, using specific Cy5-tagged monoclonal antibodies (mAbs) to target the proteins. Fig. 1 shows a complete measurement sequence starting with a bright-field image of two fixed T cells (Fig. 1A) and finishing with a high-resolution NSOM image (Fig. 1C,D) mapping the distribution of IL15R in the plasma membrane. From the confocal image shown in Fig. 1B, it is apparent that IL15R is distributed over the whole cell. Fig. 1C shows the NSOM image obtained from the region highlighted in Fig. 1B, while Fig. 1D shows with greater detail the distribution of individual receptors on the membrane with a spatial resolution of 90 nm. The high spatial resolution and surface sensitivity of NSOM provides greater detail than confocal microscopy; for example, fluorescence spots arising from single molecules (or small clusters) are readily detected on the cell surface (indicated in Fig. 1D). Additionally, fluorescent patches were also observed, indicating the presence of larger-scale clusters of IL15R on FT7.10 cells.
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The spatial organization of IL2R was also studied by NSOM in a manner similar to that used for IL15R. Fig. 2 shows a combined image of four spatially different NSOM measurements mapping the entire distribution of IL2R in the plasma membrane of an FT7.10 cell. Supplementary material Fig. S1 shows the four independent NSOM images. Just like IL15R, the cell membrane exhibits two types of protein coverage – clustered regions of IL2R displayed as bright fluorescent patches, as well as low-coverage areas containing single-molecule fluorescent spots with a size determined by the NSOM aperture and indicating the presence of only a few proteins.
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and IL15R domains and IL15R in the plasma membrane, we performed detailed quantitative analysis of the domain properties for both types of proteins. All fluorescent spots in the NSOM images of IL2R and IL15R were analyzed in terms of their intensity, as described in the Materials & Methods section. Fig. 3A,B show the intensity distributions of individual fluorescence spots of IL2R or IL15R extracted from cells labeled with Cy5-tagged 7G7 B6 or Cy5-tagged 7A4 24 mAbs, respectively. Some of the fluorescent spots were attributed to the emission of individual molecules, as they showed one-step photobleaching and a unique emission dipole moment (data not shown), signatures that are characteristic of single-molecule fluorescence detection (van Hulst et al., 2000). A typical count rate for individual Cy5 molecules of 4000 counts/second at the applied excitation intensity of 200 W/cm2 was obtained. This value was used to normalize the intensity axis on the distributions shown in Fig. 3A,B and thus to obtain an estimate of the number of Cy5 molecules (and so the number of -chains) contained in each domain.
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Interestingly, the intensity distribution of IL2R showed two main contributions: a narrow low-intensity part peaking at 1.5 Cy5 molecules (see also the inset in Fig. 3A) and a second broader-intensity distribution peaking at 130 Cy5 molecules per domain but extending up to more than 1000 Cy5 molecules per domain (Fig. 3A). These distinct distributions were, in fact, already apparent in Fig. 2, where both high-intensity patches as well as low-intensity spots were mapped. Making the reasonable assumptions that no unbound Cy5 molecules are present on the cell surface and that a one-to-one antibody-to-protein ratio is used, the lower-intensity distribution can then be attributed to individual IL2R proteins. From the analysis of seven different NSOM images, 1.4±1.1% of IL2R subunits are spatially distributed as monomers, whereas the remainder are clustered in domains. Similarly, the intensity distribution of IL15R also shows two populations, although positioned much more closely to each other. The peak values of the two populations are 3 and 15 Cy5 molecules per domain (Fig. 3B). Considering that the dye-protein labeling efficiency for IL15R is 3 (see Materials and Methods), the lower-intensity peak corresponds most probably to monomeric IL15R and accounts for 29±14% of the overall IL15R population, whereas the remaining subunits are organized as clusters on the cell membrane. Furthermore, the peak values of the intensity distributions corresponding to clustering indicated that IL2R and IL15R domains consist typically of 90 and 5 proteins, respectively. These results are fully in line with our previous flow cytometry data reporting on the significantly higher expression of IL2R in comparison with IL15R on FT7.10 cells (Vámosi et al., 2004).
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and IL15R domains and IL15R domains. The size of a domain was defined as the area within a contour line drawn by software around twice the intensity of the background level. The distribution of the occupied areas and the sizes of the IL2R and IL15R domains are shown in Fig. 4. The typical area of the IL2R domains was 0.15 µm2, corresponding to a diameter of 450 nm. Similarly, the IL15R domain areas were typically 0.10 µm2, corresponding to a diameter of 360 nm. Interestingly, while the domain sizes for IL2R and IL15R were found to be rather similar to each other, their intensities differed by more than one order of magnitude, as disclosed by the fluorescence intensity analysis (see above), reflecting a different packing density of both receptor subunits on the membrane.
To elucidate the domain packing density, we correlated the intensity versus the area of all individual domains (Fig. 5A). The correlation plot for IL2R and IL15R follows a linear dependence, indicating that the domains have a constant molecular packing density regardless of the specific domain size. In the case of the IL2R domains, the slope of the curve renders a molecular density of 1350 IL2R/µm2, indicating that on average one IL2R molecule is present in an area of 27x27 nm2. By contrast, IL15R domains have a molecular density of 120 IL15R/µm2, indicating one IL15R molecule per area of 92x92 nm2. Thus, IL2R domains are approximately ten times denser than IL15R domains.
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and IL15R intermolecular separations for each domain, assuming a random arrangement for both types of chains within the domains. The resultant distributions are displayed in Fig. 5B. Clearly, two distinct distributions for molecular distances within IL2R and IL15R are obtained. The peak of the distributions lies at 14 nm and 50 nm for the IL2R and IL15R domains, respectively. These values correspond approximately to the formula 0.5/
a, where a is the slope derived from Fig. 5B. However, it should be noted that our previous FRET data showed at least partial homodimerization/oligomerization of both IL2R and IL15R in FT7.10 cells, which necessitates a refinement of the above picture (see Discussion).
Nanometer-scale colocalization of IL2R and IL15R domains
The relative spatial arrangement of IL2R and IL15R domains in the plasma membrane of FT7.10 cells was also investigated using dual-color excitation-detection NSOM. In good accordance with our previous results obtained using FRET and CLSM (Vámosi et al., 2004), high-resolution NSOM images of the two -chains targeted with 7A4 24 (IL15R) and 7G7 B6 (IL2R) mAbs carrying different fluorophores (Alexa-Fluor-488 and Cy5, respectively) indicated considerable colocalization between IL2R and IL15R domains (Fig. 6). Hence, the correlation coefficient over ten different imaged cells rendered a value of 0.40±0.17 (see Materials and Methods). For non-domain regions, a much lower correlation (C) value was found, i.e. C = 0.17±0.09. To exclude any possible artefact due to cell autofluorescence and/or differences in labeling efficiency, we also measured the correlation coefficient from images obtained from cells labeled with Alexa-Fluor-488–7G7-B6 and Cy5–7A4-24. The values obtained remained broadly the same as before, being 0.48±0.15 for the domain regions and 0.23±0.14 for the non-domain regions.
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-chains of the two receptor types (Waldmann et al., 2001; Vámosi et al., 2004). Recent evidence suggests that IL15R on its own can mediate signals evoked by IL15 without the contribution of the βc heterodimer (Bulanova et al., 2001). It was also demonstrated that cells bearing IL15R can present the tightly associated IL15 in trans to other cells (e.g. CD8+ T cells) expressing only the β- and c-chains (Dubois et al., 2002). Membrane-bound IL15 presented in trans to target cells was found to lead to more prolonged and persistent activation of the target cells (Sato et al., 2007; Budagian et al., 2006). On cells where all the elements of the IL2R-IL15R system are coexpressed in the same membrane domains, the proximity of the distinct -chains might alter the conformation of the β- and c-subunits in distinct ways, thereby also modulating the nature of downstream signals mediated by IL2 or IL15.
Our major focus has been to investigate the lateral organization and topological relationship of IL2R and IL15R in the plasma membrane of a CD4+ human leukemia T cell line Kit 225 FT7.10. By using CLSM on FT7.10 cells, we have recently shown that the four subunits of the IL2R-IL15R system strongly colocalize with GM1-rich membrane microdomains (lipid rafts) (Vámosi et al., 2004). Furthermore, our FRET experiments suggested that IL2R and IL15R associate with each other as well as with the β- and c-chains. Here, we have further analyzed the membrane topology of IL2R and IL15R on FT7.10 cells by NSOM. The high spatial resolution provided by NSOM bridges the gap between the resolutions of CLSM and FRET experiments (300 nm and 1-10 nm, respectively). In addition, NSOM allows single-molecule detection even in densely packed areas. As such, the use of NSOM combined with single-molecule detection sensitivity has provided new insights into the organization of these receptors.
Although clustering of IL2R and IL15R at the submicron scale has also been inferred by confocal microscopy (Vámosi et al., 2004; Vereb et al., 2000), direct analysis of cluster size and density has been lacking so far. Here, we have directly observed domains of IL2R and IL15R by fluorescent means. Our data confirmed the existence of IL2R domains of typically 450 nm in diameter (Fig. 4A), in good agreement with previous CLSM and electron-microscopy studies (Vereb et al., 2000). We also observed clustering of IL15R at this hierarchical level in the plasma membrane of FT7.10 cells. IL15R domain sizes were similar to those of IL2R domains (360 nm diameter; Fig. 4B). Nevertheless, the number of -chains in their respective domains differed by more than one order of magnitude, as disclosed by the fluorescence intensity analysis of individual fluorescence spots. The latter is in good agreement with flow cytometry measurements showing that the total number of IL2R subunits expressed by FT7.10 cells is 5-10 fold higher than that of the IL15R (Vámosi et al., 2004). Finally, our current NSOM experiments confirmed the colocalization of IL2R- and IL15R-containing membrane domains (Fig. 6). On the basis of our previous CLSM and FRET data, the formation and colocalization of IL2R and IL15R domains presumably takes place in lipid rafts of FT7.10 cells and also contains other elements of the IL2R-IL15R system (Vámosi et al., 2004; Vereb et al., 2000; Matkó et al., 2002).
Interestingly, we found that the packing densities (i.e. the number of -chains per unit area) of both IL2R and IL15R domains were constant, irrespective of their sizes (Fig. 5A). This result might imply that the domains are assembled from `building blocks' with repeated structures and composition. On the basis of FRET and CLSM studies, we previously suggested the existence of `supercomplexes' formed by the elements of the IL2–IL15R system as well as MHC (and intercellular adhesion molecule ICAM-1) molecules in lipid rafts of T cells (Vámosi et al., 2004). These supercomplexes could be potential candidates for the building elements of the domains. It is probable that the constant molecular densities of IL2R and IL15R domains are optimized for their signaling function and could thus allow signaling with similar strength for all signaling domains. It is important to mention that not all the cell-surface receptors are organized in this way. For instance, the pathogen recognition receptor DC-SIGN also forms domains at the submicron scale on immature dendritic cells, but with a rather heterogeneous packing density (de Bakker et al., 2007). It has been postulated recently that, owing to its substantial glycosylation, the structure of IL2R is relatively rigid, forming a notable entropic barrier preventing the formation of the quaternary receptor complex (Chirifu et al., 2007). We hypothesize that association with MHC glycoproteins might compensate for this entropic barrier. The latter is supported by the fact that association of the IL2R complex with MHC class I was observed previously in different human T cell types, including cell lines of T lymphoma origin as well as lymphocytes from healthy donors and colorectal carcinoma patients (Matkó et al., 2002; Bene et al., 2007).
Assuming a random distribution of the -chains, the average intermolecular separation between the mutual IL2R and IL15R subunits within the domains was 14 and 50 nm, respectively (Fig. 5B). At the same time, our former FRET experiments indicated homodimeric/oligomeric molecular association of both IL2R and IL15R in the plasma membrane of FT7.10 cells (Vámosi et al., 2004). This changes the situation such that instead of (or in addition to) single entities, presumably, dimers (or higher-order oligomers) of the -chains are distributed randomly throughout the domains, which also could produce a constant packing density (necessarily with different mutual separations). This probably holds true for IL2R, but we cannot exclude that dimerization/oligomerization of IL15R is restricted to the non-domain areas (see below).
In addition to clustering at the submicron scale, we also observed a minor fraction (1%) of IL2R located in the cell membrane as small aggregates consisting of only a few proteins or even monomers. Previous FRET experiments had already hinted at the existence of a minor fraction of IL2R subunits associated with transferrin receptors (TrfRs) (Matkó et al., 2002), which are excluded from lipid rafts (Harder et al., 1998). Our findings strongly indicate that we directly observed IL2R proteins associated with the TrfR. In contrast to IL2R, a considerable portion (30%) of IL15R subunits was found to reside outside the domains and appeared in smaller clusters or even as monomers. Although we cannot completely exclude the possibility that this high percentage of IL15R outside the domains is a consequence of overexpression, the absence of IL2R molecules outside the domains argues against the possibility that artefacts are generated by overexpression of receptors. Notably, the extent of colocalization with IL2R was significantly lower for the `non-domain' IL15R molecules than that for the clustered molecules. It is conceivable that this relatively large fraction of IL15R could be responsible for those functions of IL15 that are mediated by the interaction of IL15 with IL15R alone, including βc-independent signaling routes or trans-presentation of IL15 to neighboring cells.
Recent crystallographic data have revealed considerable similarities in the topologies of the quaternary IL15-IL15R and IL2-IL2R complexes (Chirifu et al., 2007). It has been suggested that IL2, like IL15, might be capable of being presented in trans by IL2R on one cell to another cell expressing the β and the c chains. Indeed, it has been shown previously that native IL2R-bearing cells augmented the proliferative response evoked by IL2 in ex vivo large granular lymphocytic leukemia cells expressing the β and c chains but lacking the -subunits of the IL2 receptor (Eicher and Waldmann, 1998). Taking into account the rather low affinity and high off-rate of IL2R for IL2 (Liparoto et al., 2002), this process could be prominent only in cells displaying highly elevated level of the chains (e.g. numerous T lymphoma/leukemia cells, including the Kit 225 cells used in this study). Accumulation of IL2R into domains disclosed by our current and previous experiments (Vámosi et al., 2004) could further increase the avidity of trans-interactions by concentrating a large number of IL2R molecules in restricted areas and therefore enhancing the density of IL2-IL2R complexes.
Taken together, our experiments provide direct data on the size and finer structure of the large-scale (submicron size) domains of IL2R and IL15R revealed previously by confocal microscopy. Moreover, we demonstrate that, in addition to the aforementioned domains, IL15R (and a minor fraction IL2R) also exists as monomers or small clusters in the plasma membrane of T cells. Although our previous confocal and FRET experiments did not show evidence for rearrangement of IL2 and IL15 receptors on the membrane after cytokine stimulation and/or ligand binding, NSOM could potentially bring further insight into their relative organization on an intermediate spatial scale (50-200 nm). Similarly, dual-color NSOM experiments, based on the ones described here, could be performed with the aim of mapping the potential association of these receptors with lipid rafts and/or with MHC molecules. Our current and earlier data on the spatial organization of the IL2 and IL15 receptors should contribute to our understanding of the regulation of T cell functions by these cytokines. The importance of this question is also underlined by the progress towards IL2R- and IL15R-targeted immunotherapy of human leukemia, autoimmune disorders and HTLV-I-associated diseases (Waldmann et al., 2001).
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). FT7.10 cells express constitutively all subunits of the IL2R-IL15R system (Vámosi et al., 2004). In addition to the endogenous nonfunctional form of IL15R, manifesting a deletion of exons 3 and 4, FT7.10 cells have been transfected with the gene encoding the complete IL15R subunit together with an N-terminal FLAG tag. In agreement with our previous findings, cell-surface expression of the transfected IL15R was >tenfold higher than that of endogenous IL15R (Vámosi et al., 2004). The cell line was cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin and streptomycin at 37°C, in a humidified 5% CO2 atmosphere. It is important to note that the Kit225 FT7.10 cell line requires IL2 for its growth. Therefore, we also added 500 pM human recombinant IL2 to the medium every 48 hours. Cells cultured for 2 days in this medium were harvested, processed for the experiments and then analyzed, as explained below, with no extra IL2 being added before the experiments. This essentially means that the experiments were performed in an `unliganded' state. The culture medium contained 800 µg/ml G418 (Gibco) in order to suppress the growth of nontransfected wild-type cells.
Monoclonal antibodies
IL2R was targeted with mAb 7G7 B6 (IgG2a) defining an epitope that does not compete with the cytokine-binding site (Rubin et al., 1985). IL15R was labeled with 7A4 24 (IgG2), recognizing both the transfected and the native deletion mutant population of IL15R (Dubois et al., 2002; Vámosi et al., 2004). The specificity of the antibodies was proven previously by using human cell lines positive or negative for their respective antigens (Dubois et al., 2002; Yang et al., 2003). Furthermore, our flow cytometric experiments failed to detect significant binding of isotype-matched antibodies (see supplementary material Figs S2 and S3). Aliquots of purified mAbs were conjugated with succinimidyl esters of Alexa-Fluor-488 (Molecular Probes) or Cy5 (Amersham Pharmacia) dyes, as described by Szöllösi et al. (Szöllösi et al., 1996) and Sebestyen et al. (Sebestyen et al., 2002). The dye-to-protein ratio was determined spectrophotometrically. The obtained ratios were: 3 for Cy5–7A4-24 (IL15R), 2 for Cy5–7G7-B6 (IL2R), 3 for Alexa-Fluor-488–7A4-24 (IL15R) and 1 for Alexa-Fluor-488–7G7-B6 (IL2R). The fluorescent antibodies retained their affinity according to competition with identical unlabeled antibodies. To avoid possible aggregation of the antibodies, they were centrifuged at 90,000 rpm, for 30 minutes before labeling.
Labeling cells with fluorescent markers
Freshly harvested cells were washed twice in ice-cold PBS (pH 7.4). The cell pellet was suspended in PBS (0.5-1x106 cells/50 µl) and incubated with 50 µg/ml fluorescent mAbs for 45 minutes on ice. The excess of mAbs was at least 30-fold above the Kd during the incubation. Special care was taken to keep the cells at an ice-cold temperature to avoid induced aggregation or internalization of interleukin receptors. After being washed with excess cold PBS, cells were layered onto a glass coverslip and fixed with 4% formaldehyde-PBS for 60 minutes on ice, and then were dehydrated in an ethanol series (Nagy et al., 2001).
Combined confocal and near-field scanning optical microscopy
The experiments were performed using a combined confocal–near-field optical microscope equipped with single-molecule detection sensitivity (Garcia-Parajó et al., 2005). In confocal mode, incoming circularly polarized light was reflected by a dichroic mirror and focused onto the sample using an oil-immersion objective (Olympus, 64x, 1.4 NA). In NSOM mode, the light was coupled into an Al-coated tapered fiber probe (single mode, 
=633 nm, Cunz, Frankfurt), with an end-face aperture of 90 nm in diameter. The probe was kept in the near-field region of the sample (<10 nm) by means of shear-force feedback, providing simultaneously a topographic map of the sample surface while scanning (Koopman et al., 2004). A flipable mirror mount (Newfocus) enabled easy switching between excitation modes.
Single-color fluorescence experiments of Cy5-labeled proteins were performed using the 647 nm excitation line of an Ar-Kr laser at an excitation intensity of 200 W/cm2. For dual-color experiments, samples were labeled simultaneously with Alexa-Fluor-488- and Cy5-conjugated mAbs against the two -chains, avoiding possible FRET between the two fluorophores. For simultaneous excitation at 488 and 647 nm in the confocal mode, a dual-band dichroic mirror (XF2041, Omega Optical) was placed in the excitation path. The polarization and excitation intensities were adjusted independently before overlapping the two beams. Dual-color NSOM excitation was achieved by coupling the 488 nm and 647 nm lines (excitation power controlled independently for each beam) into the back-end of the fiber probe. The subwavelength aperture probe guarantees perfect overlay of the two different excitation wavelengths, without chromatic aberrations. On the detection side, the emitted fluorescence was collected by the objective and separated into two orthogonal polarization components by a broadband beam splitter (400-700 nm, Newport, Fountain Valley, CA) in the case of single-color experiments or into two spectrally separated channels for dual-color experiments and focused onto two avalanche photodiodes (SPCM-100, EG&G, Quebec) after appropriate filtering.
Cells were first screened for their overall structure and height, using the bright-field mode of the microscope. An area of typically 20x20 µm2 from the selected cell was imaged in confocal mode with either single- or dual-wavelength excitation. Regions of interest (typically 5x5 µm2) were then imaged by NSOM using single- or dual-color excitation. The same NSOM aperture probe (90 nm in diameter) was used in all experiments. In total, seven cells of the two different sample sets have been investigated by performing 30 near-field measurements with single- and dual-wavelength excitation.
Image analysis
Unprocessed images were analyzed using custom-made software that determines the size and brightness of each fluorescent spot. In the case of single-molecule spots, their size was determined using the full-width-at-half-maximum (FWHM) of a two dimensional Gaussian fit to the intensity profile. In the case of domains, their size was defined as the area within a contour line drawn by software around twice the intensity of the background level. The brightness of each spot was defined as the background-corrected sum over all pixels within the FWHM for single-molecule spots or within a contour of twice the intensity of the background level for the larger fluorescence patches.
To describe the extent of spatial overlap between IL2R and IL15R domains, cross-correlation analysis was performed on Cy5 and Alexa-Fluor-488 images obtained simultaneously from double-labeled cells. For a pair of images x and y, the cross-correlation coefficient (C) between the intensity distributions of cell-surface labeling was calculated as:
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) 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