Transmembrane cytoskeletal modulation in preterminal growing axons. II.Limax flavus agglutinin-induced receptor redistribution, capping and internalization in varicosities of growing axons

Abstract

Growing retinal ganglion cell axons of the goldfish exhibit varicosities of varying sizes and smaller non-protruding phase-dense inclusions that are mobile and mediate rapid bulk redistribution of axoplasm. In fixed axons, Limax flavus agglutinin, a lectin specific for sialic acid which has been shown to inhibit organelle transport in these axons, preferentially labels surface membrane associated with varicosities and inclusions in preterminal axons. In viable axons, Limax flavus agglutinin causes: (1) agglutination of closely apposed axons, (2) redistribution of lectin-binding sites on varicosities to surfaces of interaxonal contact with other varicosities, forming 'fused' multivaricosity complexes, and (3) formation of vacuoles in many single varicosities and some multivaricosity complexes. Vacuoles contain Limax flavus agglutinin binding sites distributed circumferentially. On the basis of immunocytochemistry, actin, myosin, calmodulin and alpha-spectrin are co-localized with redistributed Limax flavus agglutinin binding sites. The agglutination, redistribution of lectin binding sites and changes in the cytoskeleton can be reversed by treatment with sialic acid. The lectin-induced vacuole formation and internalization of Limax flavus agglutinin receptors can also be blocked either by sodium azide in a glucose-free medium, or by pretreatment with cytochalasin D and indicate an energy and a cytoskeletal dependence. The Limax flavus agglutinin-induced structural rearrangements are not altered after limited digestion with pronase. Western blots after ultramicroelectrophoresis of retinal ganglion cell axons subjected to limited digestion reveal Limax flavus agglutinin labelling of bands with apparent Mr of 64 and 70 KDa. In undigested axons, some 70 KDa protein remains unextracted with Triton X-100 lysis of axonal fields, and more remains unextracted when axonal fields are pretreated with Limax flavus agglutinin before Triton lysis, suggesting increased association with the cytoskeleton in response to lectin binding. The results indicate that cross-linking of one or more sialoglycoconjugates on the surface of varicosities of preterminal growing retinal ganglion cell axons causes a constellation of transmembrane-mediated cytoskeletal and membrane changes that are akin to those described for capping in motile cells.