Left Movie: Molecular accumulations at the interface of T cells and fibroblasts are immunologic al synapses. MCC:GFP cells and AD10 T cells were incubated for 30 minutes before fixation and staining. With anti-PKCθ (in red) and anti-phosphotyrosine (pTyr, in blue ) . Movie shows a 3D reconstruction of the APC with 2 associated T cells. The redistributed MCC:I-Ek:GFP (in green) colocalizes with PKCθ and pTyr generating the white color at the interface. Cells are at different stages of immunological synapse formation (the cell on the right is a "mature" synapse, while the cell on the left is an "immature" synapse.
Right movie: 3 Dimensional rotation of the accumulated molecules at the interface of the APC and right T cell in movie 2. Movie shows MCC:I-Ek:GFP (in green), anti-PKCθ (in red) and anti-pTyr (in blue). Cyan region is the colocalization of GFP and anti-pTyr, white is a combination of all 3 stains
Association of F-actin and the immunological synapse. Movie shows 3D rotation of a T cell – APC conjugate and immunological synapse. F-actin is stained with phalloidin (red). The location of the immunological synapse is identified by GFP -tagged MHC:peptide (green) complexes, and anti-PKCθ (blue). Also visible are trogocytosed MHC-peptide complexes (green) at the distal pole of the T cell (opposite the immunological synapse) in the movie on the right.
Trogocytosis by live cell microscopy. The T cell forms an immunological synapse at the interface with the fibroblast APC (the large green spot). After synapse formation, the T cell re-initiates locomotion and ultimately dissociates from the APC. A large green membrane fragment is captured by the T cell and it remains as an intense focus after the cells dissociate, while the remaining green molecules on the APC diffuse back into the APC membrane. This is called trogocytosis. Each image is 12 seconds apart with a total duration of 20 minutes.
In this movie, 3 individual T cells form immunological synapses with the same fibroblast APC. Upon dissociation, each trogocytoses an APC membrane fragment from the synapse (green). Each of the 3 cells is identified by a pink, orange, or gray arrow.
3D rotation of a T cell showing that trogocytosed MHC:peptide molecules (green) co-localize with the kinase p56Lck (red) and total phosphorylated tyrosine (blue). This data suggests that signaling is being driven by the trogocytosed molecules.