EXPERIMENTALCELLRESEARCH 18,,243-249 (1990) A Hyaluronan-Binding Protein Shows a Partial and Temporally Regulated Codistribution with Actin on Locomoting Chick Heart Fibroblasts E. A. TURLEY,’ P. BRASSEL, ANDD. MOORE The ultrastructural distribution of a hyaluronan- binding protein (HABP) and its relationship to actin- containing microfilaments were studied with immuno- cytochemistry. Ultrastructural analysis localized HAECP to the cell coat and demonstrated that it occurred largely in cell processes where the apical surfaces were immunopositive. The eodistribution of HABP with ac- tin-containing microfilaments in cell processes was demonstrated with double immunolabeling using monoclonal antibodies to actin and monospecific, poly- olonal antibodies to HABP. Both the topological local- ization of HABP and its cytoskeletal coassociations were modulated by cells during different cellular phases. Thus, in cells exhibiting large lamellae and few actin fibrils, typical of rapidly locomoting cells, HABP codistrihuted primarily with the actin meshwork oc- curring in cell processes, although some codistrihution between the two proteins occurredover thecell body. In cells containing prominent stress fibers and less obvious lamellae, HABP was absent in cell processes but, rather, was aligned primarily along actin fibrils oocur- ring in the cell body. A functional association between HABP and the actin-containing cytoskeleton was sug- gested by the ability of cytoeholasin D to coordinately alter the distribution of HABP and disrupt its coassoci- ation with actin. As well, the addition of hyeluronan to monolayers increased the association of HABP with a Triton-insoluble cytoskeleton. The possible roles of HABP in cell motility and cytoskeletal organization are discussed. o ISSO A~&.G~ P-, I”~. INTRODUCTION Hyaluronau has been implicated in the regulation of cell locomotion, differentiation, immune recognition, and growth both iu uitro and during the processes of morphogenesis, tissue repair, angiogenesis,and tumori- genesis [l-4]. The mechanisms by which this glycosami- noglycan influences cell behavior are not well character- ized but the demonstration, via kinetic analysis, of spe- ‘To whom correspondence and reprint requests should be ad- dressed. cific cell surface receptors [5, 61 suggests that it exerts at least part of its effects at the cell membrane. Several hyaluronan-binding proteins that represent candidates for these receptors have been isolated [7-121. One char- acterized protein, present in focal adhesions of fibro- blasts [7], has been implicated in cell adhesion and has recently been shown to associate with actin [13], raising the possibility that hyaluronan, like severalother matrix molecules [14], mediates some of its biological effects by affecting cytoskeletal organization. Another antigeni- tally distinct hyaluronan-binding protein (HABP) de- scribed by us [12] occurs predominantly on locomoting fibroblasts, where it is localized in the ruffles, blebs, and lamellae of cell processes [15]. With time after subcul- ture, this protein is lost from the cell surface and its es- duced expression coincides with declining cell locomo- tion in culture [ 161. This correlation aswell as the ability of hyaluronan to promote the motility of HABP-en- riched cells [17] suggestsa role for this protein in cell locomotion. Its regulated expression and localization suggestthat any role in mediating such behavior is dis- tinct from that of the other fibroblast, cell-associated hyaluronan-binding site [13,18]. The recent demonstration that HABP, partially puri- fied from detergent extracts of cell monolayers, contains a hyaluronan-stimulated protein kinase 1191together with the observation that hyaluronan promotes phos- phoinositide catabolism in HABP-containing cells [20] raises the possibility that this polymer may modify cell motility, at least in part, by activating messenger mecha- nisms. This possibility has precedence in the observa- tions that both protein tyrosine phosphorylation and phosphoinositide catabolism have been linked to the regulation of cytoskeletal changesthat occur during che- motactic responsesto a variety of ligands [21]. To begin to ascertain whether HABP/hyaluronan interactions might influence cytoskeletal organization during cell lo- comotion via these or other as yet undefined mecha- nisms, the spatial and functional relationship of this protein with actin-containing filaments was examined. MATERIAL AND METHODS A. Tissue Culture Seven-day-old embryonic chick hearts were explantedin Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bo- 243