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Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755
Myosin-V, an actin-based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin-V interacts with the microtubule-based motor, kinesin (1), to form a "hetero-motor" complex on vesicles. The complex of these two motors, one microtubule-based and the other actin-based, facilitates the movement of vesicular cargo from microtubules to actin filaments. In this study we show that the globular tail fragment of myosin-V displaces native myosin-V from vesicles isolated from squid brain and blocks vesicle transport on actin filaments in axoplasm from the squid giant axon.
Myosin-V recruitment and binding to neuronal vesicles are active areas of investigation. Recent studies suggest that myosin-V is recruited to vesicles by members of the Rab family of proteins (2). Rab proteins are known to regulate intracellular trafficking, and recent studies show that they also regulate vesicle transport. Rab27a is the member of the Rab family that recruits myosin-Va to melanosomes (3, 4). Melanophilin, an activator of Rab27a, has been shown to be required for binding of Rab27a to melanosomes (5). The members of the Rab family of proteins responsible for myosin-V recruitment to neuronal vesicles in the squid nervous system have not been determined, although Rab3a is known to be associated with synaptic vesicles in squid brain (6).
To study the recruitment of the globular tail domain of myosin-V to neuronal vesicles, we used a GST-tagged fragment of the globular tail in motility and vesicle-binding experiments. The mouse GST globular tail domain (MG-GTD) fragment (plasmid was a gift from Dr. Huang) contained the glutathione S-transferase (GST)-labeled mouse AF6/Cno tail-globular-domain (Fig. 1A). The 84 kDa GST-tagged fragments were expressed in E. coli, purified on affinity columns, and used in experiments with squid brain extracts and purified vesicles.
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MG-GTD was then incubated with purified squid brain vesicles to displace native myosin-V, and therefore to block vesicle transport. Vesicles were purified by Nycodenz density gradients from clarified homogenates of squid brain prepared in Tris buffered saline (TBST). The vesicle fractions from the gradient were analyzed by SDS-PAGE and western blots. Both myosin-V and kinesin were present on these vesicles (Fig. 1C). Purified vesicle fractions were incubated for 2 h at 4 °C with the GST-MyoV-tail fragment. After incubation, vesicles were fractionated on a Nycodenz gradient; blots of the vesicle fraction showed a band for the GST-tagged tail fragment, indicating binding of the tail domain to vesicles (Fig. 1D lane 4). Blots of the vesicle pellet showed an absence of myosin-V when incubated with GST-MyoV-tail (Fig. 1D lane 2), indicating displacement of native myosin-V from the vesicles by the recombinant tail fragment. Myosin was detected on the vesicles in the control (Fig. 1D lane 1), and kinesin remained on the vesicles after displacement of myosin-V by the tail fragment (Fig. 1D lane 6).
The recombinant fragment of myosin-V was then used in motility assays to determine its effects on vesicle transport. The GST-MyoV-tail fragment (at 3.12, 6.25, 12.5, 25, and 50 µg/ml) was added to freshly extruded axoplasm (Fig. 1E) in the presence of 5 mM ATP. Purified GST was used in the control experiment. After extrusion, actin filaments, detected by fluorescence microscopy (Fig. 1F), assembled at the margins of the axoplasm, and vesicle movement on these filaments was observed by AVEC-DIC microscopy (Fig. 1E). Actin-based vesicle transport was quantified by counting the number of vesicles moving/field/min (v/f/m, motile activity) at two time points during a 1-h incubation. Motility was reduced by 39% at 6.25 µg/ml, and by 80%–90% at the higher concentrations of the globular tail fragment (Fig. 1G). These data show that the headless myosin-V fragment is an effective inhibitor of vesicle transport in cell extracts and could be used to determine the mechanism of motor recruitment to vesicles.
In summary, the recombinant globular tail fragment of myosin-V pulled down kinesin by affinity-precipitation from squid brain homogenates and displaced native myosin-V from vesicles purified from these homogenates. In addition, the tail fragment blocked vesicle transport on actin filaments that assembled at the margins of axoplasm extruded from the squid giant axon. We conclude, therefore, that inhibition of vesicle transport is due to the displacement of native myosin-V from axoplasmic vesicles. We will use the globular tail domain in future experiments to study the recruitment of myosin-V to neuronal vesicles by Rab-family proteins.
Supported by NSF Grant IBN-0131470 and MBL Shifman Award to EMP-V.
Literature Cited
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  J. J. Park and Y. P. Loh Minireview: How Peptide Hormone Vesicles Are Transported to the Secretion Site for Exocytosis Mol. Endocrinol., December 1, 2008; 22(12): 2583 - 2595. [Abstract] [Full Text] [PDF]
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 T. Togo and R. A. Steinhardt Nonmuscle Myosin IIA and IIB Have Distinct Functions in the Exocytosis-dependent Process of Cell Membrane Repair Mol. Biol. Cell, February 1, 2004; 15(2): 688 - 695. [Abstract] [Full Text] [PDF]
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 J. Delacruz, J. R. Brown, and G. M. Langford Interactions Between Recombinant Conventional Squid Kinesin and Native Myosin-V Biol. Bull., October 1, 2003; 205(2): 188 - 190. [Full Text] [PDF]
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C. J. DeSelm, J. R. Brown, R. Lu, and G. M. Langford Rab-GDI Inhibits Myosin V-Dependent Vesicle Transport in Squid Giant Axon Biol. Bull., October 1, 2003; 205(2): 190 - 191. [Full Text] [PDF]
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-GST (lanes 1-3) and shows no GST in the affinity control (beads only, lane 1), 25 kDa GST in the GST control (lane 2), and GST-tagged MG-GTD (lane 3) in the experimental. The MG-GTD affinity-precipitate was also probed with