We are in the process of developing a methodology for the study of slow axoplasmic transport (axoplasmic flow) in neuronal cells maintained in tissue culture. Although this process has been documented in vivo, by injecting radioactive amino acids at the cell body and analyzing their incorporation into axonal proteins by autoradiography, it is extremely important to document and characterize it in vitro, so that we can manipulate it in detail and analyze its mechanism, in ways impossible in the whole animal. To analyze the process, we are injecting a fluorescent analogue of tubulin, one of the major proteins transported in the slow component, into the cell body; after it is fully incorporated along the full length of the neuritic process, we irreversibly bleach the fluorophore in a limited region of the process. By following the subsequent evolution of this bleached patch out the neurite, we can follow the process of axoplasmic transport of microtubule proteins. The rate of tubulin transport is being studied in a variety of different neuron types within an organism, to attempt to establish structural and functional correlates, and with respect to differentiation and pharmacological manipulation of cultured nerve cells. Finally, a number of manipulations of such cells, including temperature series and studies of the effect of calmodulin antibody injections on the process, are being assayed in attempt to get at its mechanism. Since axonal transport, and slow axonal transport in particular, is intimately linked to axonal growth, regeneration, and degeneration, it is expected that these studies will have eventual relevance to the study of diseases that involve progressive degeneration of nerves, such as amyotrophic lateral sclerosis, and may have relevance to muscular dystrophies in general.
