GTPγS inhibits organelle transport along axonal microtubules

Abstract

Movements of membrane-bounded organelles through cytoplasm frequently occur along microtubules, as in the neuron-specific case of fast axonal transport. To shed light on how microtubulebased organelle motility is regulated, pharmacological probes for GTP-binding proteins, or protein kinases or phosphatases were perfused into axoplasm extruded from squid (Loligo pealei) giant axons, and effects on fast axonal transport were monitored by quantitative video-enhanced light microscopy. GTPγS caused concentration-dependent and time-dependent declines in organelle transport velocities. GDPβS was a less potent inhibitor. Excess GTP, but not GDP, masked the effects of coperfused GRPγS. The effects of GTPγS on transport were not mimicked by broad spectrum inhibitors of protein kinases (K-252a) or phosphatases (microcystin LR and okadaic acid), or as shown earlier, by ATPγS. Therefore, suppression of organelle motility by GTPγS was guanine nucleotide-specific and evidently did not involve irreversible transfer of thiophosphate groups to protein. Instead, the data imply that organelle transport in the axon is modulated by cycles of GTP hydrolysis and nucleotide exchange by one or more GTP-binding proteins. Fast axonal transport was not perturbed by A1F4-, indicating that the GTPγS-sensitive factors do not include heterotrimeric G-proteins. Potential axoplasmic targets of GTPγS include dynamin and multiple small GTP-binding proteins, which were shown to be present in squid axoplasm. These collective findings suggest a novel strategy for regulating microtubule-based organelle transport and a new role for GTP-binding proteins.