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Biol. Bull. 207: 163. (October 2004)
© 2004 Marine Biological Laboratory

Abstract

Role of Rab GTPases in Recruitment of Myosin- V to Vesicles of Squid Giant Axon

Sung Min You1, Clarissa Cheney2, Agnieszka Swiatecka-Urban1 and George M. Langford1

1 Dartmouth College, Hanover, New Hampshire
2 Pomona College, Claremont, California

Myosin-V, an actin-based motor, is responsible for short-range axonal/dendritic transport in neurons. Recent studies have shown that Rab GTPases are required for the recruitment of myosin-V to vesicles. However, the Rab GTPase responsible for myosin-V recruitment to axoplasmic vesicles in the squid giant axon has not been determined, although our earlier data showed Rab GTPases are required for myosin-V-mediated vesicle transport. In this report, we provide additional evidence that Rab GTPases are required for the transport of axoplasmic vesicles in the squid giant axon. To study the role of Rab GTPases in myosin-V- mediated vesicle transport, a GST-labeled Rab-GDI was used in motility assays and affinity isolation experiments. A plasmid containing full-length cDNA for Drosophila GST-Rab-GDI (a gift from Dr. C. Cheney) was expressed in E. coli. The 75 kDa GST-tagged protein was purified on a GST affinity column and used in pull-down experiments with either clarified squid brain homogenates or FLAG-tagged Rab 11a transfected HEK cell lysates. Blots of the affinity isolated fractions were probed with either anti-Rab3a antibody or anti-Flag antibody. Motility assays using AVEC-DIC microscopy were performed with GST-Rab-GDI to determine whether vesicle transport in the squid giant axon is inhibited. Motile activity at 3.0 µM GST-Rab-GDI decreased from 36 ± 5 in the control assay to 15 ± 7 v/f/m. Therefore, GST-Rab-GDI inhibited vesicle transport by 58% at this concentration. At 8.0 µM GST-Rab-GDI, motile activity decreased by 88%. These data confirm that Rab activity is required for myosin-V-mediated vesicle transport in the axon. Although the Rab-GTPase responsible for Myosin-V-mediated axoplasmic vesicle transport was not identified, pull-down experiments using Rab-GDI provided potential candidate proteins. These data are consistent with published results showing that myosin-V is recruited to vesicles by Rab GTPases.

Supported by NSF Grant IBN-0131470, Dartmouth College Richter Grant, and MBL-Shifman Award to SMY.





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