Rapid mobility of motile varicosities and inclusions containing α-spectrin, actin, and calmodulin in regeneratig axons in vitro

Abstract

Time-lapse video recording was used to investigate the bidirectional movements of motile varicosities and intervening phase-dense (IPD) inclusions associated with axons of goldfish retinal ganglion cells regenerating in vitro. In addition, analyses of fine structure and immunocytochemical distributions of α-spectrin (fodrin), actin, and calmodulin in axonal fields were undertaken. Varicosities and IPD inclusions undergo saltations in a random manner at mean rates of 0.218 and 3.33 μm/sec, respectively. Experiments involving calcium antagonists or depeletion of internal calcium stores resulted in an arrest of all intra-axonal movement, indicating that saltations of the two mobile structures are dependent on intra-axonal calcium. The predominant structure in varicosities is a large aggregation of an anastomosing, tubular, smooth endoplasmic reticulum embedded in an amorphous matrix, suggesting a form of 'packaged' cytomembranes undergoing bulk transport. IPD inclusions, presumably carrying membranes, appear to shuttle between varicosities and growth cones during axon elongation, and between growth cones and varicosities during axon retraction. α-Spectrin, acting, and calmodulin were shown by immunocytochemistry to be preferentially distributed to varicosities and IPD inclusions. The co-transport of spectrin, actin, and calmodulin with cytomembranes undergoing rapid saltations departs from reported results of radioactive labeling experiments insofar as spectrin, actin, and calmodulin are not normally associated with rapidly transported membrane components in the later studies. Possible reasons for the discrepancy are discussed. Our results suggest that spectrin and actin may play a role in the packaging and axoplasmic transport of cytomembranes concerned with plasmalemma recycling.