Assembly of Myotendinous Junctions in the Chick Embryo: Deposition of P68 Is an Early Event in Myotendinous Junction Formation

Abstract

Myotendinous junctions (MTJs) are specialized sites at the surface of muscle fibers where force is transmitted between muscle and tendon. Morphogenesis of MTJs in the latter half of embryonic chick development is characterized by formation of basement membrane, appearance of subsarcolemmal densities, association of myofibrils with the membrane, and then membrane folding. In the present investigation, the time of appearance of proteins involved in force transmission at MTJs is compared to those previously described structural specializations occurring during MTJ development. In addition, P68, a recently discovered collagen-binding protein that is shown to be identical or closely related to laminin-binding lectin is also demonstrated at MTJs, and an improved technique for P68 purification is presented. P68 accumulates on the surface of collagen fibers that lie near the ends of myotubes in chick hindlimb muscle prior to the appearance of ultrastructurally discernible basement membrane. At Day 10, the stage when MTJ basement membrane is first discernible by electron microscopy, laminin and the β1 subunit of integrin are detectable at the MTJ. At this stage, laminin distribution coincides with P68 distribution and no laminin is detectable at sites other than the forming MTJs. Fibronectin is detectable at the MTJ at Day 12, but is not enriched at the MTJ relative to other sites on the myotubes. At Day 13, the stage at which subsarcolemmal densities first appear, talin is demonstrable at the MTJ and fibronectin concentration at the MTJ is enriched. Collagen type IV cannot be discerned at the MTJ until Day 15. Thus, the earliest demonstrable specializations at sites of MTJ formation are: (1) the appearance of P68 on collagen fibers that lie at the ends of myotubes, which occurs prior to ultrastructurally discernible specializations at the MTJ and (2) the accumulation of laminin at the MTJ with a distribution identical to that of P68 at the stage of MTJ formation in which basement membrane first appears. These findings indicate that P68 and laminin may mediate early events in MTJ assembly.