Nucleotide specificity for reactivation of organelle movements in permeabilized axons

Abstract

In a permeabilized axon model, exogenous ATP can reactivate intraaxonal saltatory organelle movements (microscopically visible manifestations of fast axonal transport). We have studied the dependence of the reactivated movements on the ATP concentration and have also examined the nucleotide specificity of the reactivation. Organelle transport was visualized in isolated lobster giant motor axons using Nomarski optics and video microscopy. The axons were permeabilized with saponin, and movement was reactivated with ATP or other nucleotides. Some slight movement was seen with ATP concentrations as low as 10 microM. The velocity and frequency of the reactivated transport increased with increasing ATP concentrations up to about 5 mM. Movement was also reactivated by deoxyadenosine triphosphate, but not by AMP-PNP (a nonhydrolyzable ATP analogue), ADP, or AMP. Although other nucleotides (CTP, GTP, UTP, ITP) could reactivate transport, movement equivalent to that produced by 0.1 mM ATP was only seen with tenfold or greater concentrations of the other nucleotides. This pattern of specificity is consistent with the hypothesis that a dynein-like ATPase, rather than a myosin, is involved in fast axonal transport.

Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility

“…Directed organelle movement within cells depends on ATP (Forman et al, 1984;Schliwa, 1984), and it has been generally assumed that movement is powered by an ATPase. Since all organelles move at the same velocity along isolated microtubules, the same type of molecular motor, or "translocator" may interact with a variety of organelles (Vale et al, 1985a).…”

ATP-dependent formation and motility of aster-like structures with isolated calf brain microtubule proteins.

“…Formation of aster-like structures without preformed microtubule organizing centers raises the possibility that a similar process may contribute to microtubule organization in vivo. (2,3,5), but the mechanism of action of ATP in these processes remains unknown.…”

“…Formation of aster-like structures without preformed microtubule organizing centers raises the possibility that a similar process may contribute to microtubule organization in vivo. (2,3,5), but the mechanism of action of ATP in these processes remains unknown.It has been found recently that microtubules isolated from calf brain by three cycles of assembly and disassembly will undergo "gelation-contraction" in the presence of ATP (6).We have now examined this process by computer-assisted, video-enhanced contrast microscopy (7-9). These observations reveal the formation and motility of structures with a striking resemblance to mitotic asters and spindles.…”

“…Very little is known about the mechanism of action of cytoplasmic microtubules. A requirement for ATP has been demonstrated for anaphase B (spindle elongation) (4) and for particle movements along neuronal microtubules (2,3,5), but the mechanism of action of ATP in these processes remains unknown.…”

A novel microtubule-associated protein from mammalian nerve shows ATP-sensitive binding to microtubules.

“…First, a large body of physiological and morphological evidence indicates that transport occurs in association with microtubules (17,18) probably mediated by cross-bridges between microtubules and organelles (20,24,34). Second, studies with permeabilized axons show that vesicle movement has an absolute dependence on the hydrolysis of ATP (1,14). Because of these two characteristics it has been widely anticipated for some years that rapid transport might involve a cross-bridge cycle analogous to that found in muscle or cilia.…”