Dynamic regulation of activated leukocyte cell adhesion molecule-mediated homotypic cell adhesion through the actin cytoskeleton

Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-media ted adhesion we have investigated homotypic ALCAM-ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesio n because inibition of actin polymerization by cytochalasin D (CytD) strong ly induces homotypic ALCAM-ALCAM interactions. This induction of cell. adhe sion is likely due to clustering of ALCAM at the cell surface, which is obs erved after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after C ytD treatment. In contrast, both surface distribution and adhesion of a gly cosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to Cy tD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatme nt. This demonstrates that clustering of ALCAM is essential for cell adhesi on, whereas enhanced diffusion of ALCRM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the f reely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduce d over time, suggesting strengthening of the cytoskeleton Linkage. From the se findings we conclude that activation of ALCAM-mediated adhesion is dynam ically regulated through actin cytoskeleton-dependent clustering.