Purification of LAT-Containing Membranes from Resting and Activated T Lymphocytes

Abstract

In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T-cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question, and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combines a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin resin, allowing the purification of membranes containing the Strep-tagged molecule of interest. By keeping the membranes intact, this protocol leads to the purification of molecules physically associated with the Strep-tagged protein as well as of molecules present in the same membrane compartment: transmembrane proteins, proteins strongly associated with the membranes, and luminal proteins. The example shown herein is the purification of membrane compartment prepared from T lymphocytes expressing LAT fused to a Strep-tag.

Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes

“…Large EVs that float at high densities in iodixanol gradient (Fig 4D, fractions [7-8]), and do not efficiently pass through 0.4-lm pores ( Fig 5D) promote secretion of Th2 cytokines. Note that separation of intracellular vesicles by iodixanol gradients, using the same conditions as used here for EVs, showed the presence of several transmembrane proteins in fractions [7-10] (TGN46, TfR, CD45), which therefore also contain membrane-enclosed vesicles (Hivroz et al, 2017). We only detected MHC class II in these dense fractions by WB in some of the donors analysed.…”

“…Another group has reported different quantitative effects of sEVs (called exosomes) and lEVs (called ectosomes) released by tumour cells (Keerthikumar et al, 2015), but here we also show a difference in qualitative effects. Note that separation of intracellular vesicles by iodixanol gradients, using the same conditions as used here for EVs, showed the presence of several transmembrane proteins in fractions [7-10] (TGN46, TfR, CD45), which therefore also contain membrane-enclosed vesicles (Hivroz et al, 2017). Note that separation of intracellular vesicles by iodixanol gradients, using the same conditions as used here for EVs, showed the presence of several transmembrane proteins in fractions [7-10] (TGN46, TfR, CD45), which therefore also contain membrane-enclosed vesicles (Hivroz et al, 2017).…”

Tethering of vesicles to the Golgi by GMAP210 controls LAT delivery to the immune synapse

“…We have previously shown that LAT is present in vesicles that are recruited to the immune synapse 13 . To better characterize the content of these vesicles as well as their mechanisms of transport, we purified the vesicles containing LAT and performed a proteomic analysis of their content using a method we set up in the laboratory 39 . After mechanical disruption of LAT-deficient Jurkat T cells (JCAM-2.5) 40 expressing the chimeric LAT-twin-Strep-Tag (LAT-TST) 25 , membranes were submitted to a floatation gradient.…”

Rab6-dependent retrograde traffic of LAT controls immune synapse formation and T cell activation

“…To get a better idea of the membrane compartments containing LAT, we purify these membranes and analyze their contents using a method already described ( Hivroz et al, 2017 ). In brief (graphic summary of the process in Fig.…”