3D single-particle tracking and optical trap measurements on adhesion proteins

A three-dimensional single-particle tracking system was combined with an op tical trap to investigate the behavior of transmembrane adhesion proteins. We exploited this setup to investigate which part of the cell adhesion prot ein LFA-1 forms a connection to the cytoskeleton after binding to its ligan d ICAM-1. LFA-1 is an integrin consisting of an alpha and a beta chain. Thu s far, only the cytoplasmic tail of the beta chain is known to form a conne ction to the cytoskeleton. We investigated cells that express a mutant form of LFA-1 that lacks the complete beta cytoplasmic tail and therefore is no t thought to bind to the cytoskeleton. Interestingly, single-particle track ing measurements using beads coated with the ligand ICAM-1 indicate that th is mutant form of LFA-1 does not move freely within the cell membrane, sugg esting that LFA-1 is still connected to the cytoskeleton network. This find ing is strongly supported by the observation that LFA-1 exhibits a more dif fusive motion when the cytoskeleton network is disrupted and confirmed by t he optical trap measurements used to force the proteins to move through the membrane. Collectively, our findings suggest that the interaction of LFA-1 with the cytoskeleton cannot solely be attributed to the cytoplasmic part of the beta chain. Cytometry 36:189-194, 1999. (C) 1999 Wiley-Liss, Inc.