LOCALIZATION OF UTROPHIN AND ACICULIN AT SITES OF CELL-MATRIX AND CELL-CELL ADHESION IN CULTURED-CELLS

Aciculin is a phosphoglucomutase-related cytoskeletal protein associat ed with dystrophin and/or utrophin in various tissues and cell types. Comparison of expression patterns for aciculin, dystrophin, and utroph in in cultured cells demonstrated that aciculin is coexpressed with ut rophin, but not with dystrophin, in cultures of A7r5 smooth muscle cel ls and REF52 fibroblasts. Some other nonmuscle cells synthesized only trace levels of or no aciculin and utrophin. Aciculin was detected by immunoblotting in anti-utrophin immunoprecipitates from A7r5 and REF52 cultured cells, indicating an association between these two proteins. The aciculin-utrophin complex in fibroblasts and smooth muscle cells was mostly resistant to Triton X-100 extraction and was detected predo minantly in the Triton-insoluble fraction, enriched in actin and actin -associated proteins. By immunofluorescence both aciculin and utrophin were identified in a similar dot-like or streak-like pattern in A7r5 and REF52 cultured cells. Immunolocalization of utrophin in cultured f ibroblasts and smooth muscle cells in combination with interference re action microscopy demonstrated that utrophin staining was mostly codis tributed, but not exclusively confined to the areas of focal adhesions , sites of closest cell attachment to the substrate. Double immunostai ning of A7r5 and REF52 cells for aciculin and utrophin revealed a prec ise colocalization of both cytoskeletal proteins at focal adhesions an d along microfilaments. Costaining of cultured fibroblasts and smooth muscle cells with antibodies against utrophin and major focal adhesion components, vinculin and talin, showed that utrophin is concentrated in focal adhesions both at initial stages of cell spreading and in wel l spread cells of nearly confluent monolayers. In MCF10 breast epithel ial cells both utrophin and aciculin were localized at cell-cell adher ens-type junctions. Our data show that utrophin is a cytoskeletal comp onent of cell-matrix and cell-cell adhesions in various cultured cells . In certain cell types the aciculin-utrophin complexes may contribute to the linking actin filaments to the plasma membrane. (C) 1995 Acade mic Press, Inc.