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A more recent version of this article appeared on March 1, 2002
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Submitted on June 26, 2001
Revised on November 6, 2001
Accepted on December 12, 2001
1 Molecular and Cell Biology and Biochemistry Program, Brown University, Providence, RI 02912, and Marine Biological Laboratory, Woods Hole, MA 02543
2 Marine Biological Laboratory, Woods Hole, MA 02543, and Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
3 Molecular and Cell Biology and Biochemistry Program, Brown University, Providence, RI 02912; Department of Pathology and Medicine, Brown University, Providence, RI 02912; and Marine Biological Laboratory, Woods Hole, MA 02543
* Corresponding author. E-mail address: Elaine_Bearer{at}brown.edu.
Association of motor proteins with organelles is required for the motors to mediate transport. Since axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a ~220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this ~220 kDa protein as a non-muscle myosin II. This myosin is also present in axoplasm as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using non-muscle myosin II isotype-specific antibodies detected a single ~220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment which separates organelles from cytoskeletal components. Of the total non-muscle myosin II in axoplasm 43.2 percent co-purified with organelles in the 15 percent sucrose fraction, while the remainder (56.8 percent) was soluble and found in the supernatant. This myosin decorates the surfaces of 21 percent of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, non-muscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. This myosin therefore is likely to be, in addition to myosin V, an axoplasmic organelle motor.
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