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Marine Biological Laboratory, Woods Hole, Massachusetts 02543
Almost 20 years ago, Robert Allen and colleagues (1,2) observed in squid giant axons a relatively large number of "submicroscopic" particles moving with velocities consistent with fast axonal transport. These observations were made with video-enhanced contrast-differential interference contrast microscopy (1), a methodology which had just been developed. The particles were estimated to be 30–50 nm in diameter, and they were proposed to be anatomical correlates of small vesicles apparent in electron micrographs of Hodge and Adelman (3). We recently published additional evidence in support of this view (4). Moreover, we demonstrated with immunocytochemistry that a small fraction of these vesicles contain the delayed rectifier K+ channel. This channel is also present in the axolemma, where it underlies the repolarization phase of the nerve impulse ("action potential"; 4,5). These vesicles appear not to be targeted to the axon terminals since they do not contain synaptic vesicle proteins and are not clathrin coated (4,6). We have developed novel methodology for isolating them from axoplasm (4). The initial step used in these procedures is highlighted in this report.
The medial giant axons were dissected from squid provided by the Marine Resources Center of the Marine Biological Laboratory, and the axoplasm was extruded using standard techniques (7; Fig. 1). A small amount of buffer was added (1 µl per cm length of axon) which contained 10 mM Na acetate, 10 mM HEPES (pH 7.2) with 1M glucose, so that the osmolarity was 980 mOsm. Similar results were obtained with a buffer consisting of 440 mM K glutamate, 5 mM EGTA, and 10 mM HEPES (4). Axoplasm and buffer were immediately placed in a small, thick-walled polycarbonate centrifuge tube (0.2 ml fill volume, Beckman Instruments, Inc., Palo Alto, CA) and spun in an ultramicrocentrifuge (Sorvall RC-M120GX) for 5–6 min at 35,000 gav (Fig. 1). A typical preparation consisted of axoplasm pooled from 30 axons in lots of six to minimize the time between dissection of the axons and the centrifugation step. Centrifugation yielded approximately equal volumes of translucent materials that we refer to as residual axoplasm and clear supernatant (Fig. 1).
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The supernatant (Fig. 1) is rich in low-molecular-weight proteins, as determined by SDS–PAGE with Coomassie blue staining (Fig. 2, lane a). In particular, it contains tubulin and actin (Fig. 2; arrows 2 and 3, respectively), which was confirmed by immunoblots (not shown) with anti-tubulin and anti-actin (Calbiochem, La Jolla, CA). Neurofilament proteins were not detected in an immunoblot using the antibodies described by Grant and colleagues (8). The supernatant also contains heat shock protein (Hsc70; [9]; arrow 1 in Fig. 2), as demonstrated with an immunoblot with anti-Hsc70 (Stressgen, Victoria, BC, Canada). Of particular note is the abundance of the microtubule-based motor protein kinesin in the supernatant. This protein was not detectable with SDS–PAGE either with Coomassie blue or silver staining, but was readily apparent by immunoblot (lane b, Fig. 2; anti-kinesin [Chemicon, Temecula, CA]). We estimate that 20%–40% of the total kinesin in axoplasm is contained in the supernatant of our preparation, based on densitometer tracings of immunoblots of the supernatant and residual axoplasm. Immunoblots and single-channel recordings obtained by adding an aliquot of supernatant to one side of an artificial lipid bilayer (4) demonstrated that the supernatant also contains K+ channels. In addition to kinesin, all the other proteins (Hsc70, neurofilaments, actin, and tubulin) except for K+ channels were detected in residual axoplasm.
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Our technique provides a vesicle preparation (vesicles destined for the axolemma) that is free of one of the major contaminants of vesicle preparations: neurofilament proteins. The preparation also contains a significant amount of actin and tubulin, which we believe are not associated with the vesicles. A small fraction of the heat shock protein is bound to the vesicles (unpubl. obs.), which appears to be a key factor in further purification of the vesicles (4). This preparation may be of interest to other investigators in the cellular motility field.
We gratefully acknowledge Phil Grant for his gift of neurofilament protein antibodies.
Footnotes
1 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892. 
Literature Cited
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