Cytoskeletal architecture of reactivated crayfish axons, with special reference to crossbridges among microtubules and between microtubules and membrane organelles

Bidirectional organelle movements were observed in fresh and permeabilizedreactivated (0.02% saponin, 5 mM Mg++ ATP) walking leg axons of crayfish with video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy; and the cytoskeletal organization of those axons was studied with quickfreeze, deep-etch electron microscopy (QF,DE) to understand the structure of the microtubule (MT) domain and to determine the basic cytoskeletal structures necessary for organelle transport in vivo. Vesicles and mitochondria moved bidirectionally in the central parts of fresh or permeabilized-reactivated axons. Although the axoplasm of the fresh axon was composed of longitudinally oriented microtubules and granular materials in which membrane organelles were embedded, a network of fine strands existed in the core of the granular materials. Crossbridges between membrane organelles and microtubules were present. In the central part of reactivated axons, the cytoskeleton consisted of microtubules, highly anastomosing networks of fine strands (6.6 ± 1.4 nm in width) that crosslinked the microtubules with each other, and relatively short, straight crossbridges (25 ± 3.9 nm in length, 5.5 ± 2.1 nm in width) crosslinking membrane organelles with microtubules. It has been shown that a 270KD microtubule associated protein (MAP) could be a main component of crossbridges between MTs [Hirokawa, 1986]. Hence the dynamic conformational change of crossbridges between membrane organelles and microtubules could play an important role when membrane organelles are transported.