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Signal Transduction in T cells: Imaging Approaches

Membrane Microdomains

Membrane subdomains known as “lipid rafts” have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. We have used single molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the co-receptor CD2, the adapter protein LAT, and the tyrosine kinase Lck co-cluster in discrete microdomains in the plasma membrane of signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT, and are not maintained by interactions with actin or lipid rafts. Using a two-color imaging approach that allows tracking of single molecules relative to the CD2-LAT-Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell signaling. Interestingly, CD2 appears to be central component of these microdomains, despite ambiguity as to its role in TCR signaling.


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Figure 1 - Formation of CD2-enriched signaling domains in the activated T cell surface. Jurkat cells were transiently transfected with CD2-mRFP and the indicated GFP fusion proteins. The planes of contact between the cells and anti-TCR antibody-coated coverslips were imaged by laser scanning confocal microscopy. Yellow boxes in column 1 indicate the expanded regions shown in columns 2-4. All imaging was performed in living cells at 37° C except for the CD2 + CD45 samples, which had to be fixed and stained by immunofluorescence since expression of CD45-RO-GFP at the cell surface is very low. Primary antibodies were labeled with Zenon labeling kits (Molecular Probes) to avoid labeling of the glass-adsorbed, stimulatory antibody. Scale bars, 10 µm (whole cells) and 2 µm (expanded regions).





Figure 2 - Dual color imaging of single GFP-tagged molecules relative to CD2-mRFP clusters. (A) A single frame from an image sequence of single molecules of Lck-GFP (green) that was superimposed upon a snapshot, binarized image of CD2-mRFP clusters (red)(see Suppl. Experimental Procedures). The movie of this cell can be found in Suppl. Movie 3. Panels B-D show centroid trajectories of single molecules of Lck-GFP, LAT-GFP, and LAT(C-S)-GFP, illustrating that they alternate between periods of immobilization within CD2 clusters and more rapid mobility outside of the clusters (see also Suppl. Movies 6-9). Panels E-J show examples of several single molecule trajectories in which centroid trajectories navigate in channels between CD2 zones (see also Suppl. Movies 4, 5. In B-I, the trajectory color indicates the spatial density of single molecule centroids within a 150 nm2 neighborhood, as a means of showing spatial confinement. Scale bars: (A) 5 µm, (B-I) 2 µm. The durations of each trace in seconds, in the left and right panels, are: (B) 1.00, 2.80 s; (C) 5.17, 6.03 s; (D) 3.77, 3.80 s; (E) 3.27, 4.57 s; (F) 2.83, 3.0 s; (G) 1.27, 3.63 s; (H) 2.63, 1.90 s; (I) 1.47, 2.10 s; (J) 4.93, 2.23 s.

Movie 1 - Centroid trajectory representing a single molecule of LAT-GFP diffusing among CD2-mRFP signaling domains. A single molecule image sequence was acquired at video rate, the observed particle was tracked, and the centroid positions were then drawn over a still image of CD2-mRFP clusters in the same cell. The LAT-GFP molecule shows transient confinement behavior upon entering the CD2 domains.

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Movie 2 - Centroid trajectory representing a single molecule of Lck-GFP diffusing among CD2-mRFP signaling domains. Diffusional trapping in the CD2 regions and exclusion at their peripheries can be observed.


CD2-CD58 interaction activates microdomain formation and signaling in Jurkat T-cells in the absence of T cell receptor activation

Activation of T-cell by antigen presenting cells (APC) is thought to involve the ligation of the T cell receptor (TCR) and a subsequent tyrosine phosphorylation cascade, which includes activation of a critical scaffold protein, LAT. Another T cell surface protein called CD2, which interacts with human CD58 on the APC, also has been implicated in signaling but its role and requirement has been controversial. Through real time imaging in a reconstituted system consisting of Jurkat T-cells and a planar lipid bilayer (as a mimic of an APC), we found that activated TCR and CD2 that interacts with CD58 in the bilayer separate into distinct microdomains. Surprisingly, LAT tended to colocalize most strongly with the CD2 domains. These CD2-CD58-LAT microdomains displayed robust phosphotyrosine staining, indicating a signaling function. In addition, such phosphorylation is observed in CD2-CD58-LAT microdomains, in the absence of TCR activation. These results suggest an important role of CD2 in T-cell activation, through the organization and activation of signaling molecules in the T cell membrane.

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updated 4/9/07

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