T‐Cell Antigen Receptor‐Induced Signaling Complexes: Internalization Via a Cholesterol‐Dependent Endocytic Pathway

Abstract

T-cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP-76, detected as SLP-yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP-76 movement at 168C, this movement required endocytosis. Immunoelectron microscopy showed SLP-76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP-76 clusters, as did coexpression of the ubiquitin-interacting motif domain from eps15. These data are consistent with the internalization of SLP-76 via a lipid raft-dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP-76 clusters contained phosphorylated SLP-76 and phosphorylated LAT. The raft-associated, transmembrane protein LAT likely targets SLP-76 to endocytic vesicles. The endocytosis of active SLP-76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.

T Cell Polarity at the Immunological Synapse Is Required for CD154-Dependent IL-12 Secretion by Dendritic Cells

“…Alternatively, because CD154 is rapidly endocytosed (41), its enrichment at the IS may involve polarization of the recycling endosomal compartments toward the T cell-APC contact zone. Such a mechanism has been shown to contribute to the enrichment of TCRs, CD71, and the signaling molecule linker for activation of T cells at the IS (16,45,56). This polarized secretion of CD154 at the IS is probably crucial for CD154 signaling by nonactivated memory and naive T cells that express low levels of CD154 (43,44) and in this study (Fig.…”

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

“…To engage the TCR, cells were plated onto coverslips coated with stimulatory anti-CD3 antibodies, an experimental system that we have used extensively to study T-cell activation (Barr et al, 2006;Bunnell et al, 2001Bunnell et al, , 2002.…”

Ubiquitylation-Dependent Negative Regulation of WASp Is Essential for Actin Cytoskeleton Dynamics

“…WASp binds and activates the Arp2/3 complex and promotes actin polymerization and its recruitment to the TCR site (3,5,30). Late in the activation process, vesicles containing both SLP-76 and WASp are endocytosed via a lipid raft-dependent pathway (5,7).…”