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Biol. Bull. 201: 240-241. (October 2001)
© 2001 Marine Biological Laboratory

Recombinant Globular Tail Fragment of Myosin-V Blocks Vesicle Transport in Squid Nerve Cell Extracts

Jeremiah R. Brown, Kyle R. Simonetta, Leslie A. Sandberg, Phillip Stafford1 and George M. Langford

Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755

Myosin-V, a calmodulin-binding myosin motor, mediates the movement of vesicles on cortical actin filaments in a variety of cell types. This motor has been shown to transport ER and synaptic vesicles in neurons, melanosomes in melanocytes, and secretory vesicles and the vacuole in yeast. Recent evidence (1) suggests that the globular tail of myosin-V, which binds to the surface of vesicles (2,3), interacts with the microtubule-based motor, kinesin, to form a "hetero-motor" complex on vesicles. The complex of these two motors, one microtubule-based and the other actin-based, is thought to facilitate the movement of vesicular cargo from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons (4), we hypothesize that one of the functions of the tail-tail interaction is to provide feedback between the two proteins to allow a seamless transition of vesicles from microtubules to actin filaments.

To study the interactions of the globular tail domain of myosin-V to kinesin and to neuronal vesicles, we used a glutathione S-transferase (GST)-tagged globular tail fragment in motility and vesicle-binding experiments. The plasmid for the recombinant tail fragment of mouse myosin-V was provided by Dr. Huang (1). The plasmid contained the GST-labeled mouse AF6/cno tail-globular-domain (GST-MyoV-tail [aa1569 to aa1768] without the coiled medial tail domain). After expression in E. coli, the GST-tagged fragment was purified on affinity columns and used in experiments with squid brain extracts and purified vesicles.

The GST-MyoV-tail fragment was identified on blots with a GST antibody (Fig. 1A, lanes 4 and 5; 1C, lanes 5 and 6). To determine whether the GST-MyoV-tail fragment binds squid brain kinesin, squid brain extracts were incubated with GST-MyoV-tail fragments for 2 h at 4°C, and then the GST-labeled fragment was immunoprecipitated with the GST antibody. Blots of the proteins isolated by this immunoprecipitation (IP) showed a kinesin band when probed with the H2 antibody to squid brain kinesin (Fig. 1A, lane 3), establishing that the recombinant mouse myosin-V-tail pulled down native squid kinesin.



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  		Figure 1. (A) Western blot analyses of immunoprecipitation (IP) experiments using the GST-MyoV-tail. Clarified squid optic lobe homogenate was Triton X-100 extracted, incubated with the recombinant tail fragment for 2 h at 4°, and recovered using {alpha}-GST. (Lane 1) Squid myosin-V enriched fraction (S5B) probed with {alpha}-P196, a polyclonal squid myosin-V antibody (* denotes band of interest). (Lane 2) IP-GST-MyoV-tail probed with {alpha}-P196. (Lane 3) IP-GST-MyoV-tail probed with {alpha}-H2, a monoclonal antibody to the squid kinesin. (Lane 4) IP-GST-MyoV-tail probed with {alpha}-GST antibody. (Lane 5) Purified GST-MyoV-tail probed with {alpha}-GST. (B) Western blot analyses of sucrose vesicle fractions obtained by running clarified squid brain homogenate on a sucrose density gradient (0.3/0.6/1.5 M gradient; vesicle fraction taken from 0.3/0.6 M interface). (Lane 1) Vesicle fraction probed with {alpha}-QLLQ, a polyclonal antibody to the squid myosin-V tail. (Lane 2) Vesicle fraction probed with {alpha}-H2. (C) Western blot analyses of vesicle fraction incubation GST-MyoV-tail. The vesicle fraction, prepared by resuspending vesicular pellet from clarified squid brain homogenate, was incubated for 2 h at 4° with the GST-MyoV-tail. The control was incubated only with buffer. The supernatant and vesicle pellet were analyzed by western blot analyses. (Lane 1) Control supernatant probed with |ga-QLLQ. (Lane 2) GST-MyoV-tail incubation supernatant probed with |ga-QLLQ. (Lane 3) Control pellet probed with {alpha}-QLLQ. (Lane 4) GST-myosin-V tail incubation probed with {alpha}-QLLQ. (Lane 5) GST-MyoV-tail incubation pellet probed with {alpha}-GST. (Lane 6) Purified GST-MyoV-tail probed with {alpha}-GST. (D) GST-MyoV-tail inhibition experiments. GST-MyoV-tail was added to squid giant axon extracts at time zero. GST was added to the control. Vesicles moving/field/minute (motile activity) was measured for the control at 15 min. Motile activity was measured for the GST-MyoV-tail at 45 min. Each measured for concentrations of 0.25 mg/ml and 0.5 mg/ml. (E) The motile activity at each GST-MyoV-tail concentration compared with the control is plotted as percent (%) inhibition. Percent inhibition determined by comparing 15-minute control time point with the 45-minute experimental time point.

 
The bacterially expressed recombinant globular tail domain of myosin-V was incubated with purified squid brain vesicles to replace native myosin-V. Vesicles were purified by sucrose density gradient from clarified homogenates of squid brain. Vesicle fractions were examined by DIC and fluorescence microscopy after staining with DIOC6, a green fluorescent membrane dye. Overlay of the DIC and fluorescent images demonstrated that the particles observed in the DIC image were membrane structures. The vesicle fractions were analyzed by SDS-PAGE and western blots, and both myosin-V and kinesin were present on these vesicles (Fig. 1B, lanes 1 and 2). A similar vesicle fraction was incubated for 2 h at 4°C with the GST-MyoV-tail fragment. After incubation, blots of the vesicle pellet showed a band for the GST-tagged fragment, indicating binding of the tail domain to vesicles (Fig. 1C, lane 5). Blots of the supernatants, after the vesicles were pelleted, showed a higher concentration of myosin-V in the GST-MyoV-tail incubation than in the control incubation, indicating displacement of native myosin-V from the vesicles by the recombinant tail (Fig. 1C, lanes 1 and 2).

The recombinant fragment of myosin-V was used in motility assays to determine whether it had replaced native myosin-V on axoplasmic vesicles and blocked transport. The GST-MyoV-tail fragment (0.25 and 0.5 mg/ml) was added to axoplasm in the presence of 5 mM ATP, and the sample was warmed to 24°C. Purified GST was used as a control. Actin-based vesicle transport was quantified by counting the number of vesicles moving/field/min (v/f/m, motile activity) at 2 time points during a 1-h incubation. Motile activity for the 0.25 mg/ml trial decreased from 17.5 ± 5.5 to 2.6 ± 1.3 v/f/m, and for the 0.5 mg/ml trial, from 16.7 ± 1.7 to 1.5 ± 0.5 v/f/m (Fig. 1D). Therefore, the MyoV tail fragment inhibited vesicle transport by 81% and 91%, respectively, and thereby exhibited a dominant negative effect in these functional assays (Fig. 1E). These data show that the recombinant protein blocked the activity of native myosin-V presumably by binding to vesicles and competing away the native myosin-V motors.

The GST-MyoV-tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates, and it therefore exhibited binding properties of native myosin-V. The GST-MyoV-tail fragment blocked vesicle transport in extracts of the squid giant axon. These data show that the headless myosin-V fragment is an effective inhibitor of vesicle transport in cell extracts and can be used to determine the mechanism of motor recruitment to vesicles. These studies support the hypothesis that tail-tail interactions may be a mechanism for feedback between myosin-V and kinesin, allowing transition of vesicles from microtubules to actin filaments.

Supported by NSF grant MCB9974709 and MBL Josiah Macy Fellowship.

Footnotes

Motorola, Inc. Chicago, IL Back

Literature Cited

  1. Huang, J. D., S. T. Brady, B. W. Richards, D. Stenolen, J. H. Resau, N. G. Copeland, and N. A. Jenkins. 1999. Nature,397:267–270.[Medline]
  2. Wu, X., B. Bowers, K. Rao, Q. Wei,J. A. Hammer III. 1998. J. Cell Biol., 143:1899–1918.[Abstract/Free Full Text]
  3. Reck-Peterson, S. L., P. J. Novick, and M. S. Mooseker. 1999. Mol. Biol. Cell, 10:1001–1017.[Abstract/Free Full Text]
  4. Tabb, J. S., B. J. Molyneaux, D. L. Cohen, S. A. Kuznetsov, and G. M. Langford. J. Cell Sci.111:3221–3234.



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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.
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