GTP gamma S inhibits organelle transport along axonal microtubules.

Abstract

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. GTP3,S caused concentration-dependent and time-dependent declines in organelle transport velocities. GDP/3S was a less potent inhibitor. Excess GTP, but not GDP, masked the effects of coperfused GTP3'S. The effects of GTP3,S 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 ATP3,S. Therefore, suppression of organelle motility by GTP3,S 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 GTP'yS-sensitive factors do not include heterotrimeric G-proteins. Potential axoplasmic targets of GTP3,S include dynamin and multiple small GTPbinding 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.

Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal Neurons

“…Shorter and slower moving tubulovesicular post-Golgi structures have been described in neurons (Nakata et al, 1998) and unpolarized cells (Hirschberg et al, 1998;Toomre et al, 1999). Axonal transport in different systems was reported to be between 1 and 3 m/s on average, sometimes with maximal velocities of up to 5 m/s (Allen et al, 1982;Kreis et al, 1989;Bloom et al, 1993;Lochner et al, 1998;Nakata et al, 1998;Zakharenko and Popov, 1998;Bridgman, 1999). The velocity measured for APP-YFP in our system is in the range of the fast component of fast axonal transport in mammals in vivo, which has been calculated to have a velocity of 410 mm/d (Ochs, 1972), corresponding to 4.7 m/s.…”

1-Methyl-4-phenylpyridinium affects fast axonal transport by activation of caspase and protein kinase C

“…However, these experiments did not identify specific PKC isoforms involved. Previous studies ruled out pathways mediated by conventional, calcium-and DAG-activated PKCs (PKC␣, ␤, and ␥) (62). The absence of a plasma membrane in the isolated axoplasm model similarly eliminates the possibility that membrane receptormediated signaling pathways are required for activation of PKC.…”

“…However, the PKC kinase family consists of 12 or more serine/threonine-specific isoenzymes (42), and the identity of PKC isoforms mediating MPPϩ effects remained to be determined. The conventional PKCs (␣, ␤, and ␥) could be ruled out because increased Ca 2ϩ and DAG do not affect FAT (43). Among the novel and atypical PKCs, PKC␦ is a well established substrate of caspases (44,45).…”

The Mechanism for Regulation of the F-actin Binding Activity of IQGAP1 by Calcium/Calmodulin

“…With one modification, protocols described previously were also used for quantitation of GTP overlay blots (24), gel electrophoresis (5), and immunoblotting (5). The modification was to use a Umax (Freemont, CA) Astra 2200 scanner with a transparency adapter and 12-bit grayscale depth, instead of a CCD camera, to capture digital images of gels and chemiluminescent Western blots.…”