Identification of molecules in a muscle extracellular matrix extract that promotes process outgrowth from cultured adult frog motoneurons.

Abstract

The molecular composition of the substrate on which neurons are cultured is critical for their attachment, survival, and extension of processes. The aim of the present experiments was to characterize the molecules in an extracellular matrix (ECM) extract that promotes the outgrowth of processes from cultured adult frog motoneurons. An extract was made of skeletal muscle ECM and tested as a substrate for cultured motoneurons. The average total process length of motoneurons cultured on this crude ECM extract is greater than when the neurons are cultured on concanavalin A, poly-L-lysine or mouse tumor (EHS) laminin. Gel filtration of the ECM extract yielded fractions with an increased specific activity for promoting process outgrowth. The most active fractions exhibit a single major polypeptide band of ca. 1 mD and two minor bands of ca. greater than 1 mD and 205 kD upon sodium dodecyl sulfate gel electrophoresis. Under reducing conditions, three major bands were seen of 340, 205, and 200 kD. Electron microscopy of rotary-shadowed ECM fractions showed macromolecules with a cross-shaped structure similar to vertebrate and invertebrate laminin, a rod-like molecule resembling vertebrate and invertebrate collagen type IV, and a third molecule similar in appearance to vertebrate fibrillin. These results represent the first step in analyzing the role of substrate molecules in promoting neuromuscular reinnervation.