LIGAND-BINDING TO HEPARAN-SULFATE PROTEOGLYCANS INDUCES THEIR AGGREGATION AND DISTRIBUTION ALONG ACTIN CYTOSKELETON

Cell surface heparan sulfate proteoglycans (HSPGs) participate in mole cular events that regulate cell adhesion, migration, and proliferation . The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HS PGs and their distribution along underlying actin filaments. Immunoflu orescence and confocal microscopy and immunogold and electron microsco py indicate that, in the absence of ligands, HSPGs are irregularly dis tributed on the fibroblast cell surface, without any apparent codistri bution with the actin cytoskeleton. In the presence of ligand (lipopro tein Lipase) or antibodies against heparan sulfate, HSPGs aggregate an d colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSP Gs were clustered and associated with the actin filaments. Cross-linki ng experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surf ace. These cross-linked proteoglycans appeared in the Triton X-100 ins oluble fraction. Platinum/carbon replicas of the fibroblast surface in cubated either with lipoprotein lipase or antiheparan sulfate showed l arge aggregates of HSPGs regularly distributed along cytoplasmic fiber s. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were inc ubated either with Lipoprotein lipase or antibodies against heparan su lfate, the distance between immunofluorescence spots was uniform. In c ontrast, the spacing between HSPGs on fixed cells not incubated with l igand was more variable. This highly organized spatial relationship be tween actin and proteoglycans suggests that cortical actin filaments c ould organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by hepa rin-binding proteins.