Membrane lipid order of sub‐synaptic T cell vesicles correlates with their dynamics and function

During an immune response, T cells survey antigen presenting cells for antigenic peptides via the formation of an interface known as an immunological synapse. Among the complex and dynamic biophysical phenomena occurring at this interface is the trafficking of sub‐synaptic vesicles carrying a variety of proximal signalling molecules. Here, we show that rather than being a homogeneous population, these vesicles display a diversity of membrane lipid order profiles, as measured using the environmentally sensitive dye di‐4‐ANEPPDHQ and multi‐spectral TIRF microscopy. Using live‐cell imaging, vesicle tracking and a variety of small molecule drugs to manipulate components of the actin and tubulin cytoskeleton, we show that the membrane lipid order of these vesicles correlate with their dynamics. Furthermore, we show that the key proximal signalling molecule Linker for Activation of T cells (LAT) is enriched in specific vesicle populations as defined by their higher membrane order. These results imply that vesicle lipid order may represent a novel regulatory mechanism for the sorting and trafficking of signalling molecules at the immunological synapse, and, potentially, other cellular structures.