Single-molecule tracking of membrane molecules: plasma membrane compartmentalization and dynamic assembly of raft-philic signaling molecules.

Tracking single molecules in the plasma membrane in live cells is becoming a useful technique for studying the spatial-temporal control of membrane molecular processes, such as signal transduction and the formation of large molecular complexes. In this review, three topics largely based on recent single-molecule observations are described, with a special emphasis on the results that are considered to be difficult to obtain using conventional methods monitoring the ensemble-averaged behavior of molecules. First, we describe the high-speed single-molecule tracking data, mostly obtained by our group that necessitated the paradigm shift of the plasma membrane structure, from the two-dimensional continuum fluid model to the compartmentalized fluid model. Second, we try to present a synthetic view of the cell membrane, which contains raft and other microdomains as well as being partitioned into small compartments. Furthermore, we present our working hypothesis, based on the literature, how large, stabilized rafts may be formed, after ligation or crosslinking, from small/unstable "reserve" rafts present in the steady-state cells. Finally, we explain our initial application of single-molecule fluorescence imaging for studies of the creation of T-cell receptor signaling complexes (immunological synapses or SMACS), by observing the recruitment of single Lck molecules as an initial approach. This revealed that the assembly of Lck at the T-cell receptor cluster site, observed by conventional fluorescence microscopy, actually represents dynamic concentrations of Lck molecules, entering and exiting the cluster domain rapidly, with the aid of the raft domains.