Alpha 5 integrin is a critical component of adhesion plaques in myogenesis.
We investigated the distribution and expression of alpha 5 beta 1 and alpha 3 beta 1 integrin in differentiating myogenic cells in culture. The myogenic cells expressed both alpha 5 and alpha 3 integrin subunits with the same molecular sizes as those expressed by chicken embryo fibroblasts (CEF). However, the ratio of total alpha 5 to alpha 3 was threefold higher in the muscle cultures than that in CEF cultures. A new method is described whereby adhesion plaque-associated integrin was cross-linked to its extracellular matrix ligand on the substrate using a nonpenetrating cross-linker, BS3, and integrin not involved in substrate adhesion as well as cytoskeletal proteins were removed with a zwitterionic detergent. alpha 5 and beta 1 integrin, but not alpha 3 could be cross-linked to fibronectin at adhesion plaques throughout myogenesis in culture. Alpha 5 beta 1 integrin was found only at the edge of myoblasts 4 hr after plating but became distributed under their entire surface by 1 day in culture. When the muscle cells became elongated, a morphology they express after the initiation of terminal differentiation, and as they began to fuse, alpha 5 was found redistributed in small adhesion plaques along the lateral edges of the postmitotic myocytes and early myotubes. In mature myotubes, which are large multinucleated branched structures, alpha 5 beta 1 integrin was localized to parallel streaks underneath their entire substrate surface. Throughout the different stages of myogenesis, vinculin colocalized with alpha 5 beta 1 integrin in adhesion plaques, but alpha-actinin only colocalized to the adhesion plaques in myoblasts, not in myotubes. These studies suggest that alpha 5 beta 1 integrin through its dynamic interaction with both fibronectin and the cytoskeleton is important for both the signals which initiate the differentiation process and for subsequent morphological and structural changes during the differentiation process.
Enomoto
M I
MI
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6076.
Boettiger
D
D
Menko
A S
AS
D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation; D002478:Cells, Cultured; D002642:Chick Embryo; D003432:Cross-Linking Reagents; D005109:Extracellular Matrix / Q000502:physiology; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D016023:Integrins / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D009132:Muscles / Q000196:embryology*; D017469:Receptors, Fibronectin / Q000378:metabolism*; D015398:Signal Transduction
D003432:Cross-Linking Reagents; D016023:Integrins; D007797:Laminin; D017469:Receptors, Fibronectin