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Biol. Bull. 205: 188-190. (October 2003)
© 2003 Marine Biological Laboratory

Interactions Between Recombinant Conventional Squid Kinesin and Native Myosin-V

John Delacruz, Jeremiah R. Brown and George M. Langford

Dartmouth College, Hanover, NH

Axoplasm from the squid giant axon is one of a small number of cell-free extracts within which axonal transport can be studied in vitro (1). In squid axoplasm, one can observe both microtubule-based and actin-based vesicle transport and the seamless transition of vesicles from microtubules to actin filaments (2). Based on studies of vesicle transport in this cell-free preparation, a new model of axonal transport has emerged called "dual transport" in which long-range vesicle transport is microtubule-based while short-range transport is actin-based (3).

An exciting recent discovery is the finding that the cargo-binding domains of myosin-V, an actin-based motor, and kinesin, a microtubule-based motor, interact to form a hetero-motor complex (4, 5, 6, 7). The interaction of myosin-V with kinesin has been established through yeast 2-hybrid assay, co-immunoprecipitation, co-affinity isolation, and co-purification with myosin-V (4, 5, 6, 7). The distal/globular tail domain of myosin-V binds to the rod-tail domain of kinesin in the "hetero-motor" complex. The members of the kinesin super family that have been shown to bind to myosin-V include conventional or ubiquitous kinesin (kinI) and Smy1p (4, 5, 6, 7). Evidence that myosin-V and kinesin interact on the membrane surface has not yet been demonstrated.

In this report, we performed experiments to determine the functional significance of the interactions between kinesin and myosin-V. Our working hypothesis is that tail-tail interactions between these motors provide feedback and thereby allow coordination of motor activity during the transition of vesicles from microtubules to actin filaments. For example, one motor may become inactive when its partner is actively transporting a vesicle along a filament (3). Several studies have shown that the ATPase activity of kinesin is inhibited when the head and tail domains of the molecule interact (8, 9). Auto-inhibition may be the mechanism by which one motor becomes inactive when its partner motor binds to a filament. Therefore, only one motor is actively engaged in movement and a tug-of-war between motors is prevented. Such feedback between motors could explain the seamless transition of vesicles from microtubules to actin filaments observed in the squid giant axon.

In this study we used a histidine-tag bound to the tail fragment of squid conventional kinesin (His-tagged) to study the interactions between kinesin and myosin-V. A cDNA construct coding for the rod-tail domain of conventional squid kinesin (SK KhcU; gift of K. Kosik) was engineered into a vector containing a His-tag. The 1.5 kb SK KhcU contained most of the rod II domain and the entire tail domain including the stop codon. The sequence of the insert was confirmed by PCR. The His-kinesin vector was expressed in E. coli, and the recombinant protein was then purified on a nickel-column. A gel of the fraction eluted from the column with 40 mM imidazole showed a prominent band at 45 kDa, the expected molecular weight of the fragment (Fig. 1A, lane 1). This band was identified as the His-labeled kinesin fragment on nitrocellulose membranes that were probed with the His-antibody (Fig. 1A, lane 2).



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  		Figure 1. (A) The presence of the His-tagged recombinant squid kinesin tail construct was verified using SDS-PAGE and western blots, and sequencing data. Lane 1 shows an SDS-PAGE gel confirming the presence of a protein with a molecular weight of 45 kDa, which matches the expected weight of the kinesin construct. Lane 2 shows a western blot probed with anti-His antibody; the blot confirms that the 45 kDa protein contains a His-tag. (B) The squid kinesin tail fragment is shown to interact with native squid myosin-V obtained from homogenized optic lobes. The homogenized extract was run through a His-kinesin affinity column. Lane 1 shows an SDS-PAGE gel of the elution fraction and shows that the 45 kDa kinesin tail and the 196 kDa native myosin-V are present. The elution fraction was blotted and probed with myosin-V antibody {alpha}-QLLQ (lane 2) and confirms myosin-V. (C) Allen Video Enhanced Contrast-Digital Interference Contrast (VEC-DIC) microscopy is used to image microtubules and vesicle motility in extruded squid axoplasm. Experiments are being performed to determine whether the recombinant kinesin tail fragment conclusively inhibits vesicle motility along microtubules.

 
Next, we applied a clarified extract of His-kinesin tail fragment to a Ni-column to make a kinesin tail affinity column. Nonspecific proteins were removed by several buffer washes; and a clarified squid optic lobe extract was then passed over the column, followed by buffer washes. The proteins that bound specifically to the tail domain of kinesin were eluted with imidazole and analyzed by SDS-PAGE. Multiple protein bands were visible on the gel of the eluted fraction (Fig. 1B, lane 1). The proteins were transferred to membrane and probed with an antibody to myosin-V, a known binding partner of kinesin. A prominent band was observed on the blot (Fig. 1B, lane 2). These data showed that the recombinant His-kinesin fragment interacted with myosin-V and several other proteins in the squid brain extract. Finally, we asked whether the His-kinesin fragment blocked microtubule-based vesicle movement in motility assays. Preliminary experiments were performed in which the His-kinesin tail fragment was added to extruded axoplasm from the squid giant axon (Fig. 1C). Vesicle transport was measured by counting the number of vesicles moving/microtubule/min (v/mt/m; motile activity) at 15-min intervals. A reduction of motile activity occurred but the number of replicates was not sufficient to provide conclusive evidence.

In summary, recombinant, His-labeled, squid kinesin tail fragment binds to squid brain myosin-V, as demonstrated by affinity column isolation. The recombinant tail fragment is thus an excellent tool for identifying specific binding partners of kinesin and potentially for studies of kinesin-mediated vesicle transport.

This work was supported by NSF Grant IBN-0131470, and the Leadership Alliance Grant and MBL-Shifman Award to JMD.

Literature Cited

  1. Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson. 1985. Cell Motil. Cytoskelet. 1: 291–302.
  2. Kuznetsov, S. A., G. M. Langford, and D. G. Weiss. 1992. Nature 356: 722–725.[Medline]
  3. Langford, G. M. 2002. Traffic 3: 859–865.[ISI][Medline]
  4. Brown, J. R., K. R. Simonetta, L. A. Sandberg, P. Stafford, and G. M. Langford. 2001. Biol. Bull. 201: 240–241.[Free Full Text]
  5. Brown, J. R., E. M. Peacock-Villada, and G. M. Langford. 2002. Biol. Bull. 203: 210–211.[Free Full Text]
  6. 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]
  7. Lillie, S. H., and S. S. Brown. 1998. J. Cell Biol. 140: 873–883.[Abstract/Free Full Text]
  8. Freidman, D. C., and R. D. Vale. 1999. Nat. Cell Biol. 1: 288–292.[ISI][Medline]
  9. Coy, D. L., W. O. Hancock, M. Wagenbach, and J. Howard. 1999. Nat. Cell Biol. 1: 288–297.




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