Assessing Immunological Synapse Topology through Live-Cell Imaging
Trascrizione
Take a microscope dish with buffer containing transfected, superantigen-treated human endothelial cells, with the superantigens complexed to class II MHC molecules.
These artificial antigen-presenting cells co-express different fluorescent proteins on their membranes and cytoplasm.
Next, introduce T lymphocytes, which interact with the superantigens on the endothelial cells via T cell receptors, forming a stable cell-cell junction, an immunological synapse.
This triggers T lymphocyte cytoskeleton rearrangement, initiating cell spreading and actin extension, resulting in the formation of invadosome-like protrusions, or ILPs, enriched with actin, adhesion, and signaling molecules.
Adhesion molecules bind specific receptors on the endothelial cells, strengthening the interaction.
Synapse formation activates T lymphocyte signaling pathways, elevating intracellular calcium levels and stabilizing ILPs.
ILPs induce localized membrane bending in endothelial cells, forming transient surface rings.
Under a fluorescence microscope, analyze the synapse topology comprising dark circular zones of fluorescent cytoplasm displacement in the endothelial cell, co-localized with differently fluorescent membrane rings around ILPs, confirming immunological synapse formation.
