Single-Molecule Microscopy Reveals Plasma Membrane Microdomains Created by Protein-Protein Networks that Exclude or Trap Signaling Molecules in T Cells

Abstract

Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, 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 coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster 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.

Lipid rafts: contentious only from simplistic standpoints

“…The conclusion of Larson et al 41 is that a protein-interaction domain, provided by the activated kinase domain of Lyn, is required for stable association with the crosslinked-IgE receptor. A similar conclusion is equally plausible in the case of interactions between activated LCK with the TCR-signalling domain 31 . Analysis of the microlocalization of the minimal anchors of Lyn and LCK and the cognate full-length proteins has largely relied on detergent insolubility with all its inherent problems (BOX 2), except for a FRET study that showed cholesterol-dependent clustering of the minimal Lyn anchor 29 .…”

“…Analysis of the microlocalization of the minimal anchors of Lyn and LCK and the cognate full-length proteins has largely relied on detergent insolubility with all its inherent problems (BOX 2), except for a FRET study that showed cholesterol-dependent clustering of the minimal Lyn anchor 29 . The new studies 31,41 show that the surface distribution of the minimal anchor and the full-length proteins are different, and therefore illustrate the importance of protein-protein as well as anchor-lipid-bilayer interactions in controlling the stable association of proteins with L o domains.…”

“…These basic properties of the model predict that cluster formation is cholesterol-dependent, but do not predict or require the existence of stable, pre-existing, large L o domains. Larger, even more stable domains can be formed, but these require proteinprotein interactions and/or crosslinking -as with the creation of T-cell-signalling domains 31 . Note that this model is different from the previously advanced lipid-shell hypothesis 32 , which suggests that lipid shells that are formed around lipid anchors operate as targeting motifs that allow interaction of the shelled protein with pre-existing lipid rafts or caveolae 32 .…”

“…In an elegant set of SFVT experiments examining formation of T-cell receptor (TCR) signalling platforms, Douglass and Vale (REF. 31 ) showed that full-length lymphocyte-specific protein tyrosine kinase (LCK) stably interacts with CD2-positive and linker for activation of T cells (LAT)-positive TCR-signalling domains, but that the minimal lipid anchor of LCK coupled to GFP does not. Similarly, Larson et al 41 showed that full length Lyn co-localizes (actually codiffuses as this is an FCS analysis) with crosslinked immunoglobulin E (IgE) receptor, but again the minimal lipid anchor of Lyn coupled to GFP does not.…”

Lipid rafts, cholesterol, and the brain

“…Protein diffusion during T-cell signaling, another example of a signaling event thought to involve lipid rafts, has also been investigated using single molecule techniques. This approach revealed an unexpected role of protein-protein networks in regulating the enrichment or exclusion of proteins from sites of T-cell signaling (Douglass and Vale 2005).…”