Localization of hyaluronate and hyaluronate-binding protein on motile and non-motile fibroblasts.

The distribution of a hyaluronate-binding (HABP) and rhodamine B-isothiocyanate (RITC)-labeled hyaluronate (HA) were studied on both actively motile and stationary chick heart fibroblasts to assess the relationship of these molecules to each other, to other extracellular matrix molecules, to membrane protrusions and to adhesion sites. RITC-HA and HABP, detected by indirect immunofluorescence, were concentrated in the perinuclear region, the leading lamella and retraction processes of actively motile cells, although RITC-HA also occurred diffusely over the rest of the cell body. Double immunofluorescence confirmed that HA and HABP co-localized in the former three regions, suggesting that, at these locations, the HABP may act as a cell surface-binding site for HA. With increasing culture confluency and consequent slowing of fibroblast motility, the localization of both polymers changed to a uniform and diffuse distribution over the cell body and processes. On actively motile cells, RITC-HA and HABP did not co-distribute with fibronectin, heparan sulfate proteoglycan or laminin. Areas coated with RITC-HA and HABP often contained specialized adhesion sites as determined by interference reflection microscopy (IRM) but neither polymer appeared to particularly localize to adhesion sites. However, the occurrence of RITC-HA and HABP in the leading lamellae of motile cells consistently coincided with ruffling activity. These results are discussed with respect to a possible instructive role of HA in cell motility.