Localization of utrophin and aciculin at sites of cell-matrix and cell-cell adhesion in cultured cells.
Aciculin is a phosphoglucomutase-related cytoskeletal protein associated with dystrophin and/or utrophin in various tissues and cell types. Comparison of expression patterns for aciculin, dystrophin, and utrophin in cultured cells demonstrated that aciculin is coexpressed with utrophin, but not with dystrophin, in cultures of A7r5 smooth muscle cells and REF52 fibroblasts. Some other nonmuscle cells synthesized only trace levels of or no aciculin and utrophin. Aciculin was detected by immunoblotting in antiutrophin 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 predominantly 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 fibroblasts and smooth muscle cells in combination with interference reflection microscopy demonstrated that utrophin staining was mostly codistributed, but not exclusively confined to the areas of focal adhesions, sites of closest cell attachment to the substrate. Double immunostaining of A7r5 and REF52 cells for aciculin and utrophin revealed a precise colocalization of both cytoskeletal proteins at focal adhesions and 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 well spread cells of nearly confluent monolayers. In MCF10 breast epithelial cells both utrophin and aciculin were localized at cell-cell adherens-type junctions. Our data show that utrophin is a cytoskeletal component 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.
Belkin
A M
AM
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090, USA.
Burridge
K
K
D008841:Actin Cytoskeleton / Q000737:chemistry; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000032:analysis*; D005347:Fibroblasts / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D007365:Intercellular Junctions / Q000737:chemistry*; D008565:Membrane Proteins; D009130:Muscle, Smooth / Q000166:cytology; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D010733:Phosphoglucomutase; D051381:Rats; D049411:Utrophin
D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; C484256:Pgm5 protein, rat; C483600:Utrn protein, mouse; D049411:Utrophin; D010733:Phosphoglucomutase