The role of kinesin and other soluble factors in organelle movement along microtubules.
Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to participate in microtubule-based organelle transport, the exact role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine the ability of soluble factors to promote organelle movement. Salt-washed organelles from squid axoplasm exhibited a nearly undetectable level of movement on purified microtubules. Their frequency of movement could be increased greater than 20-fold by the addition of a high speed axoplasmic supernatant. Immunoadsorption of kinesin from this supernatant decreased the frequency of organelle movement by more than 70%; organelle movements in both directions were markedly reduced. Surprisingly, antibody purified kinesin did not promote organelle movement either by itself or when it was added back to the kinesin-depleted supernatant. This result suggested that other soluble factors necessary for organelle movement were removed along with kinesin during immunoadsorption of the supernatant. A high level of organelle motor activity was recovered in a high salt eluate of the immunoadsorbent that contained only little kinesin. On the basis of these results we propose that organelle movement on microtubules involves other soluble axoplasmic factors in addition to kinesin.
D000818:Animals; D000911:Antibodies, Monoclonal; D001369:Axons / Q000166:cytology; D001685:Biological Factors / Q000502:physiology*; D001692:Biological Transport; D049832:Decapodiformes; D003720:Densitometry / Q000379:methods; D066298:In Vitro Techniques; D016547:Kinesins; D008870:Microtubules / Q000502:physiology; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000502:physiology*; D015388:Organelles / Q000378:metabolism / Q000502:physiology*; D011193:Potassium Iodide / Q000494:pharmacology; D012995:Solubility
D000911:Antibodies, Monoclonal; D001685:Biological Factors; D009419:Nerve Tissue Proteins; D011193:Potassium Iodide; D016547:Kinesins