Cutting edge: cell surface linker for activation of T cells is recruited to microclusters and is active in signaling

Abstract

A controversy has recently emerged regarding the location of the cellular pool of the adapter linker for activation of T cells (LAT) that participates in propagation of signals downstream of the TCR. In one model phosphorylation and direct recruitment of cell surface LAT to activation-induced microclusters is critical for T cell activation, whereas in the other model vesicular, but not surface, LAT participates in these processes. By using a chimeric version of LAT that can be tracked via an extracellular domain, we provide evidence that LAT located at the cell surface can be recruited efficiently to activation-induced microclusters within seconds of TCR engagement. Importantly, we also demonstrate that this pool of LAT at the plasma membrane is rapidly phosphorylated. Our results provide support for the model in which the cell utilizes LAT from the cell surface for rapid responses to TCR stimulation.

Figure 1. Characterization of CD4-LAT

A. Schematic of CD4-LAT chimeric construct. B. JCam2.5 cells stably expressing CD4-LAT were transiently transfected with LAT-YFP. Cells were then dropped onto stimulatory coverslips, permeabilized and immunostained for CD4. Images 2 microns above the coverslip (top panel) or at the coverslip (bottom panel) are shown. Pearson’s Correlation Coefficient values averaged over 6 cells ± S.E. are indicated to the right of the images. C. JCam2.5 cells or cells stably transduced with CD4-LAT were stimulated with 10μg/ml OKT3 and assayed for intracellular calcium flux.

Figure 2. Cell surface LAT is recruited to microclusters

A. JCam2.5 cells stably expressing CD4-LAT or CD4-LAT41 were labeled with anti-CD4-488 (clone OKT4) at 4°C, dropped onto stimulatory coverslips at 37°C and fixed after 2 minutes. Post-fixation, cells were permeabilized and immunostained for phosphotyrosine. Confocal slices 0.7μm apart were collected through the entire cell. B. JCam2.5 cells stably expressing CD4-LAT were labeled with anti-CD4-488 at 37°C and imaged as above. Lower panel: Cells labeled at 37°C were stripped of surface antibody by low pH rinse. Following washing in buffer at neutral pH, cells were dropped onto stimulatory coverslips and imaged. C. JCam2.5 cells stably expressing CD4-LAT were labeled with anti-CD4-488 at 4°C, dropped onto stimulatory coverslips and imaged live by TIRF microscopy. Still images 84 seconds apart are shown from Movie 1. D. JCam2.5 cells stably expressing CD4-LAT or CD4-LAT41 were transiently transfected with Grb2-YFP, dropped onto stimulatory coverslips at 37°C, fixed after 2 minutes and immunostained for CD4 without permeabilization. Pearson’s Correlation Coefficient values are indicated to the right of the images.

Figure 3. Cell surface LAT is phosphorylated

A. JCam2.5 cells stably expressing CD4-LAT or CD4-LAT41 were labeled with anti-CD4-488 (clone OKT4) at 4°C, dropped onto stimulatory coverslips at 37°C and fixed after 2 minutes. Post-fixation, cells were permeabilized and immunostained for pLAT¹⁹¹. Confocal slices 0.7μm apart were collected through the entire cell. B. JCam2.5 cells stably expressing CD4-LAT were either unlabeled (no biotin) or labeled with sulfo-NHS-biotin at 4°C (4°C label). After quenching and washing, cells were stimulated for 1 minute at 37°C, lysed, and biotinylated proteins were isolated on neutravidin-agarose beads. Whole cell lysates (lysate) and affinity purified samples (AffP: neutravidin-agarose) were then immunoblotted as indicated. C. Lysates from unlabeled or biotinylated cells were subjected to 3 rounds of serial purification on neutravidin-agarose beads. Input and post-purification lysates (Post 3X AffP) were immunoblotted as indicated. D. Three models for LAT recruitment to signaling microclusters. LAT can move laterally within the PM to be recruited to microclusters. In this model activated ZAP-70 phosphorylates LAT at the PM (1). LAT in microclusters could be recruited from vesicles that originate from a recycling pool from the plasma membrane (2) or directly from the Golgi apparatus (3). In models 2 and 3, ZAP-70 phosphorylates vesicular LAT in trans. In this paper we provide evidence for model 1 at early time-points after activation. At the right, a microcluster is internalized and directed towards degradation pathways.