The cytoskeleton in myelinated axons: a freeze-etch replica study.

The organization of the cytoskeleton in myelinated axons of the rat has been analyzed without chemical fixation in replicas of deep-etched materials after rapid freezing. Freeze-etch replicas of trigeminal nerves provided three-dimensional views of the well-developed cytoskeleton inside axons. In these preparations, the axonal cytoskeleton was seen to be composed of longitudinally-oriented microtubules and neurofilaments which were interconnected by slender strands. Such strands also connected membranous organelles with microtubules and neurofilaments. After Triton X-100 extraction, the neurofilament-associated interconnecting strands (cross-linking filaments) persisted, indicating that they are not artifactual products of soluble protein condensation during freeze-etching. In non-extracted axons many granular structures were closely associated with cytoskeletal components. These granular elements were not seen after Triton treatment. These findings, together with fluorographic analyses, suggest that the granular structures may represent. These findings, together with fluorographic analyses, suggest that the granular structures may represent slowly transported "soluble proteins' in axoplasm. This freeze-etch replica study, without any chemical fixation, substantiates the reality of the axonal cytoskeleton which is directly involved in the axonal transport. Furthermore, using this approach ultrastructural evidence was obtained of the close association of membranous structures with the cytoskeleton.