Recent evidence on the occurrence of small (5-700 nm diameter) lipid microdomains in the exoplasmic leaflet of the plasma membrane has evoked interest in the possibility that similar domains may also be present in the cytoplasmic leaflet of the plasma membrane. However, current knowledge about these "lipid rafts", in live cells is limited. One way to obtain insight into the occurrence and the size of lipid rafts is the use of single-molecule microscopy, which allows one to study the diffusive motion of individual molecules with high positional and temporal accuracy. Using this technique, we compared the diffusion behavior of the Lck membrane anchor, which has a high affinity for lipid rafts, to the diffusion behavior of the K-Ras membrane anchor, which has negligible affinity for rafts and compared the results with those of the H-Ras membrane anchor. Surprisingly, we found only minor differences in the diffusion behavior of the various lipid anchors, indicating that putative cytoplasmic leaflet lipid rafts would have to be small (<137 nm diameter) and do not affect the mobility of membrane-anchored molecules much on timescales up to 60 ms.

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Supramolecular clusters at the immunological synapse provide a mechanism for structuring complex communication networks between cells of the immune system. Regulating intra-and intercellular trafficking of proteins and lipids to and from the immunological synapse provides an additional level of complexity in determining the functional outcome of immune cell interactions. An emergent principle is that molecules requiring tightly regulated cell surface expression, e.g. negative regulators of cell activation or molecules promoting cytotoxicity, are trafficked to the immunological synapse from intracellular secretory lysosomes as required. Many molecules required for the early stages of the intercellular communication are already present at the cell surface, sometimes in lipid rafts, and are rapidly translocated laterally to the intercellular contact. Our understanding of these events critically depends on utilizing appropriate technologies for probing supramolecular recognition in live cells. Thus, we also present here a critical discussion of the technologies used to study lipid rafts and, more broadly, a map of the spatial and temporal dimensions covered by current live cell physical techniques, highlighting where advances are needed to exceed current spatial and temporal boundaries.