Video-enhanced contrast-differential interference contrast microscopy has revealed new features of axonal transport in the giant axon of the squid, where no movement had been detected previously by conventional microscopy. The newly discovered dominant feature is vast numbers of "submicroscopic" particles, probably 30- to 50-nanometer vesicles and other tubulovesicular elements, moving parallel to linear elements, primarily in the orthograde direction but also in a retrograde direction, at a range of steady velocities up to +/- 5 micrometers per second. Medium (0.2 to 0.6 micrometer) and large (0.8 micrometer) particles move more slowly and more intermittently with a tendency at times to exhibit elastic recoil. The behavior of the smallest particles and the larger particles during actual translocation suggests that the fundamental processes in the mechanisms of organelle movement in axonal transport are not saltatory but continuous.

Development of video-enhanced contrast-differential interference contrast for light microscopy has permitted study of both orthograde and retrograde fast axonal transport of membranous organelles in the squid giant axon. This process was found to continue normally for hours after the axoplasm was extruded from the giant axon and removed from the confines of the axonal plasma membrane. It is now possible to follow the movements of the full range of membranous organelles (30-nanometer vesicles to 5000-nanometer mitochondria) in a preparation that lacks a plasma membrane or other permeability barrier. This observation demonstrates that the plasma membrane is not required for fast axonal transport and suggests that action potentials are not involved in the regulation of fast transport. Furthermore, the absence of a permeability barrier surrounding the axoplasm makes this an important model for biochemical pharmacological, and physical manipulations of membranous organelle transport.

Native microtubules prepared from extruded and dissociated axoplasm have been observed to transport organelles and vesicles unidirectionally in fresh preparations and more slowly and bidirectionally in older preparations. Both endogenous and exogenous (fluorescent polystyrene) particles in rapid Brownian motion alight on and adhere to microtubules and are transported along them. Particles can switch from one intersecting microtubule to another and move in either direction. Microtubular segments 1 to 30/~m long, produced by gentle homogenization, glide over glass surfaces for hundreds of micrometers in straight lines unless acted upon by obstacles. While gliding they transport particles either in the same (forward) direction and]or in the backward direction. Particle movement and gliding of microtubule segments require ATe and are insensitive to taxol (30/~M). It appears, therefore, that the mechanisms producing the motive force are very closely associated with the native microtubule itself or with its associated proteins.Although these movements appear irreconcilable with several current theories of fast axoplasmic transport, in this article we propose two models that might explain the observed phenomena and, by extension, the process of fast axoplasmic transport itself. The findings presented and the possible mechanisms proposed for fast axoplasmic transport have potential applications across the spectrum of microtubule-based motility processes.

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The development of AVEC-DIC microscopy and the application of this method to the study of fast axonal transport in isolated axoplasm extruded from the giant axon of the squid Loligo pealei provides a new paradigm for analyzing the intracellular transport of membranous organelles. The size of the axon, the number of transported particles, and the absence of permeability barriers like the plasma membrane in this preparation permit many experiments that are difficult or impossible to perform using other model systems. The use and features of this preparation are described in detail and a number of properties are evaluated for the first time. The process of extrusion is characterized. Particle movement is evaluated both in the interior of extruded axoplasm and along individual fibrils that extend from the periphery of perfused axoplasm. The role of divalent cations, particularly Ca2+, and the effects of elevated Ca2+ on axoplasmic organization and transport are analyzed. A series of pharmacological agents and polypeptides that alter cytoskeletal organization are used to examine the role of microfilaments and microtubules in fast transport. Finally, the effects of depleting ATP and of adding ATP analogues are discussed. The extruded axoplasm preparation is shown to be an invaluable model system for biochemical and pharmacological analyses of the molecular mechanisms of intracellular transport.