RESUMEN
The existence of lipid rafts and their importance for immunoreceptor signaling is highly debated. By non-invasive single molecule imaging, we analyzed the dynamics of the T-cell antigen receptor (TCR), the lipid raft-associated glycosylphosphatidylinositol (GPI) proteins CD48 and CD59 and the major leukocyte phosphatase CD45 in living naive T lymphocytes. TCR triggering induced the immobilization of CD45 and CD48 at different positions within the T-cell interface. The second GPI protein, CD59, did not co-immobilize indicating lipid raft heterogeneity in living T lymphocytes. A novel biochemical approach confirmed that lipid raft components are not associated in the plasma membrane of resting cells, and variably associate with specific receptors to distinct lipid rafts upon activation.
Asunto(s)
Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Receptores de Antígenos de Linfocitos T/ultraestructura , Linfocitos T/inmunología , Antígenos CD/metabolismo , Antígenos CD/ultraestructura , Complejo CD3/metabolismo , Complejo CD3/ultraestructura , Antígeno CD48 , Antígenos CD59/metabolismo , Antígenos CD59/ultraestructura , Membrana Celular/química , Membrana Celular/ultraestructura , Glicosilfosfatidilinositoles/química , Humanos , Cinética , Antígenos Comunes de Leucocito/metabolismo , Antígenos Comunes de Leucocito/ultraestructura , Activación de Linfocitos , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Microscopía Confocal , Movimiento (Física) , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Linfocitos T/ultraestructuraRESUMEN
In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS. This distribution of ezrin and CD43 at the inhibitory NK cell IS is similar to that previously seen at the activating T cell IS. CD45 is also excluded from the inhibitory, but not activating, NK cell IS. In addition, electron microscopy reveals wide and narrow domains across the synaptic cleft. Target cell HLA-C, located by immunogold labeling, clusters where the synaptic cleft spans the size of HLA-C bound to the inhibitory killer Ig-like receptor. These data are consistent with assembly of the NK cell IS involving a combination of cytoskeletal-driven mechanisms and thermodynamics favoring the organization of receptor/ligand pairs according to the size of their extracellular domains.