Endocytic events in TCR signaling: focus on adapters in microclusters

Abstract

Although the critical role of T-cell receptor (TCR) microclusters in T-cell activation is now widely accepted, the mechanisms of regulation of these TCR-rich structures, which also contain enzymes, adapters, and effectors, remain poorly defined. Soon after microcluster formation, several signaling proteins rapidly dissociate from the TCR. Recent studies from our laboratory demonstrated that the movement of the adapters linker for activation of T cells (LAT) and Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) away from initial microcluster formation sites represents endocytic events. Ubiquitylation, Cbl proteins, and multiple endocytic pathways are involved in the internalization events that disassemble signaling microclusters. Several recent studies have indicated that microcluster movement and centralization plays an important role in signal termination. We suggest that microcluster movement is directly linked to endocytic events, thus implicating endocytosis of microclusters as a means to regulate signaling output of the T cell.

Fig. 1. SLP–YFP internalizes in uncoated structures

Jurkat T cells expressing SLP–YFP were plated onto stimulatory coverslips, fixed after incubation at 37 °C for 5 min, stained with anti-GFP antibodies, and processed for electron microscopy. Enhanced gold staining is seen on uncoated pits, vesicles, and internal membranes. Arrowheads indicate pits and vesicles; Arrows indicate tubules, e-endosomes, G-Golgi, c-cap shaped structures similar to recycling endosomes. Insert shows higher magnification view of pits budding from the plasma membrane. Bar (main panel) = 280 nm, bar (insert) = 170 nm. Originally published in Traffic 2006;7:1143–1162, Publisher Wiley-Blackwell.

Fig. 2. Persistent LAT clusters caused by over expression of 70Z/3 Cbl contain phosphorylated LAT

Jurkat T cells expressing LAT–YFP were transfected with either 70Z/3 Cbl–CFP (top row) or wildtype (WT) Cbl–CFP (bottom row). Twenty-four hours after transfection, the cells were plated onto stimulatory coverslips and fixed after incubation at 37 °C for 5 min. The cells were stained with mouse anti-phosphotyrosine (4G10) and rabbit anti-phospho-LAT¹⁹¹. LAT–YFP is shown in the green channel, anti-phosphotyrosine staining in the red channel, anti-phospho-LAT¹⁹¹ staining in cyan, and Cbl–CFP in yellow. In the cells expressing 70Z/3 Cbl-CFP, there are prominent clusters containing both LAT–YFP and 70Z/3 Cbl–CFP that show extensive staining for phosphotyrosine and phospho-LAT. In contrast, cells overexpressing WT Cbl–CFP have faint LAT clusters, no Cbl–CFP clusters, very little phosphotyrosine staining, and almost undetectable phospho-LAT staining. Bar = 5 μm.