Assay of vesicle motility in squid axoplasm

Axoplasm prepared as described above will maintain high levels of fast axonal transport for 1-2 hours, although moderate decrements in the average velocity may be noted over time. The organelles and structures that can be detected in isolated axoplasma are as small as the 50-nm synaptic vesicles or 25-nm microtubules, well below the limits of resolution for light microscopy; however, in the center of the axoplasm, where the density of structures is high, individual microtubules are not readily distinguished and individual vesicles can be followed only for short distances before they move out of the plane of focus or are lost in the multitude of neighboring organelles. On the periphery of perfused axoplasm, each of these structures may be readily detected and analyzed, but some information is lost about the role of specific axoplasmic organization in normal transport processes. Fortunately, the juxtaposition in one preparation of essentially structurally intact axoplasm with the extracted individual microtubules transporting organelles provides a unique preparation for molecular dissection of intracellular transport (Brady et al., 1985).