The lateral mobility of the neural cell adhesion molecule (NCAM) was examined using single particle tracking (SPT). Various isoforms of human NCAM, differing in their ectodomain, their membrane anchorage mode, or the size of their cytoplasmic domain, were expressed in National Institutes of Health 3T3 cells and C2C12 muscle cells. On a 6.6-s time scale, SPT measurements on both transmembrane and glycosylphosphatidylinositol (GPI) anchored isoforms of NCAM expressed in 3T3 cells could be classified into mobile (Brownian diffusion), slow diffusion, corralled diffusion, and immobile subpopulations. On a 90-s time scale, SPT studies in C2C12 cells revealed that 40-60% of transfected NCAM was mobile, whereas a smaller fraction (approximately 10-30%) experienced much slower diffusion. In addition, a fraction of approximately 30% of both transfected GPI and transmembrane isoforms and endogenous NCAM isoforms in C2C12 cells experienced transient confinement for approximately 8 s within regions of approximately 300-nm diameter. Diffusion within both these and the slow diffusion regions was anomalous, consistent with movements through a dense field of obstacles, whereas diffusion outside these regions was normal. Thus the membrane appears as a mosaic containing regions that permit free diffusion as well as regions in which NCAM is transiently confined to small or more extended domains. These results, including a large, freely diffusing fraction, similar confinement of transmembrane and GPI isoforms, a significant slowly diffusing fraction, and relatively large interdomain distances, are at some variance with the membrane skeleton fence model (Kusumi and Sako, 1996). Possible revisions to the model that incorporate these data are discussed.
