Report |
Address correspondence to Vladimir I. Gelfand, Dept. of Cell and Structural Biology, University of Illinois at Urbana-Champaign, 601 S. Goodwin Ave., Rm. B107. Urbana, IL 61801. Tel.: (217) 333-5927. Fax: (217) 244-1648. E-mail: vgelfand{at}life.uiuc.edu
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Abstract |
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Key Words: kinesin II; dynactin; dynein; melanophores; organelle transport
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Introduction |
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Whereas very little is known about how kinesin II associates with cargo organelles, this issue has been extensively characterized for cytoplasmic dynein. It has been demonstrated that dynein associates with many of its cargoes through the dynactin complex (Karki and Holzbaur, 1999). Dynactin is a complex of at least 10 polypeptides that range in size from 24 to 150 kD (Schroer, 1996). The best characterized subunits of dynactin are p150Glued and p50 (dynamitin).
p150Glued contains both microtubule-binding and dynein-binding domains. p150Glued binds microtubules through the NH2-terminal CAP-Gly motif (Vaughan and Vallee, 1995; Waterman-Storer et al., 1995), and phosphorylation near this motif has been shown to regulate microtubule binding (Vaughan et al., 2002). p150Glued interacts directly with the intermediate chain of cytoplasmic dynein (DIC) (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995), and this interaction is thought to be essential for dynein-mediated organelle transport (Gill et al., 1991; Holzbaur et al., 1991; Holleran et al., 1998; King and Schroer, 2000). Regulation of this interaction by DIC phosphorylation suggests an important function (Pfister et al., 1996; Vaughan et al., 2001).
Dynamitin is localized in the shoulder region of the complex and is thought to hold p150 and the rest of the complex together (Schroer, 1996; Eckley et al., 1999). Exogenous levels of dynamitin disrupt the dynactin complex and provide a tool to study dynactin function (Echeverri et al., 1996).
Mechanisms of kinesin II cargo recognition can be investigated using Xenopus melanophores, which contain hundreds of pigment organelles (melanosomes). These cells can be stimulated to aggregate melanosomes to the cell center by melatonin, which reduces the concentration of intracellular cAMP or disperse them by melanocyte stimulating hormone (MSH), which increases intracellular cAMP (Daniolos et al., 1990). Aggregation is accomplished by cytoplasmic dynein (Nilsson and Wallin, 1997), whereas dispersion requires the combined action of myosin V and kinesin II (Rogers and Gelfand, 1998; Tuma et al., 1998; Gross et al., 2002a). The identification of kinesin II as the microtubule motor responsible for melanosome dispersion and the ability to activate pigment dispersion facilitates functional studies of kinesin II in melanophores, making them an ideal system to study this motor.
In this report, we have investigated the role of dynactin in bidirectional melanosome transport. This work demonstrates that p150Glued and the KAP subunit of Xenopus kinesin II interact and that this interaction is required for melanosome dispersion. In addition, our results show that p150Glued does not bind XKAP and the DICs at the same time, suggesting a novel regulatory mechanism to control the direction of motility.
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Results and discussion |
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Visual observations of these cells indicated that melanosomes ceased to move either toward or away from the cell center. We then determined the percentage of melanosomes undergoing directed movements, which were defined as any linear melanosome movement with a distance of at least 2 µm, at a rate of at least 0.2 µm/s. This analysis (Table I) demonstrates that overexpression of EGFPdynamitin strongly suppressed both anterograde and retrograde components of organelle movement, suggesting that a disruption of the dynactin complex inhibits both dynein- and kinesin IIbased transport. At the same time, staining with a tubulin antibody showed that overexpression of dynamitin did not result in depolymerization of microtubules, and only a small fraction of transfected cells (<5%) showed microtubule arrays that were not tightly focused at the centrosome (unpublished data).
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Competition between kinesin II and dynein for binding to p150Glued
The region of p150Glued implicated in kinesin II binding was also sufficient to bind the cytoplasmic dynein intermediate chain. This raised the question of whether dynactin could bind kinesin II and dynein simultaneously. To assess this possibility, immunoprecipitations were performed using polyclonal antibodies against the 95-kD subunit of kinesin II and DIC (Fig. 3 A). Both kinesin II and DIC were able to pull out p150Glued. However, no kinesin II was detected in antidynein immunoprecipitates and vice versa, suggesting that the two motors cannot bind to p150Glued at the same time.
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Functional analysis of the kinesin IIdynactin interaction
The data presented above demonstrates that disruption of the dynactin complex inhibits both retrograde and anterograde melanosome transport and that kinesin II and dynactin physically interact. If dynactin is truly required for bidirectional melanosome transport, then blocking the DICXKAP binding site on p150Glued should inhibit both anterograde and retrograde transport.
To test this hypothesis, we measured organelle movements in cells overexpressing XKAP or its COOH- and NH2-terminal fragments. Overexpression of XKAP greatly reduced both anterograde and retrograde melanosome transport (Table I) with no detectable change in microtubule distribution (unpublished data). Furthermore, this inhibition was observed in cells transfected with C-XKAP, confirming that the binding of XKAP to p150Glued is mediated by the COOH-terminal portion of XKAP (Table I). Cells transfected N-XKAP showed some inhibition of anterograde but not retrograde melanosome transport, possibly by binding one or both of the motor subunits of kinesin II. These results provide functional confirmation to the suggestion that kinesin II utilizes p150Glued as a binding platform on the melanosome surface.
The results presented here demonstrate that p150Glued interacts with both kinesin II and dynein on melanosomes. The identification of the KAP subunit as being responsible for the interaction of kinesin II with other proteins agrees with the findings of several different studies. Yeast two-hybrid analysis demonstrated that KAP interacts with a member of the Wnt signaling pathway, adenomatous polyposis coli, and is required for adenomatous polyposis coli localization to the tips of growing microtubules (Jimbo et al., 2002). Other KAP binding partners have been identified in this way, including Smg-GDS (Shimizu et al., 1996), XCAP (Xenopus chromosomeassociated polypeptide) (Shimizu et al., 1998), and nonerythroid spectrin (Takeda et al., 2000). Conversely, it was shown that p150Glued interacts with the mitotic kinesin, HsEg5, suggesting that dynactin may interact with members of the kinesin superfamily other than kinesin II (Blangy et al., 1997).
Our results for the first time provide physical evidence to the theory that anterograde and retrograde motility of organelles on microtubules is coordinated. This idea was first put forth by Schroer et al. (1988), who showed that immunodepletion of conventional kinesin inhibited both plus and minus end directed vesicle transport. A similar result was obtained when antibodies against conventional kinesin inhibited bidirectional vesicle movement in axoplasm (Brady et al., 1990). Waterman-Storer et al. (1997) demonstrated that inhibitory antibodies against p150Glued blocked the motility of organelles obtained from extruded squid axoplasm along microtubules in both directions. Indications of motor coordination were also observed in genetic studies with Drosophila. Welte et al. (1998) demonstrated that a mutation in the klarsicht gene inhibited microtubule transport of lipid droplets in both directions. In addition it was shown that mutations in the gene encoding Drosophila Glued inhibited plus enddirected vesicle transport in addition to dynein based transport (Martin et al., 1999; Gross et al., 2002b). The inhibition of anterograde and retrograde vesicle movement was also observed in fibroblasts transfected with dynamitin (Valetti et al., 1999). Exactly how the coordination of anterograde and retrograde microtubule motors is accomplished remains to be seen, but this report demonstrates that for cytoplasmic dynein and kinesin II the inhibition of one motor can lead to inhibition of the opposite polarity motor as a result of both motors sharing the same binding component on the cargo surface.
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Materials and methods |
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Molecular biology and transfection
pEGFPdynamitin was a gift from R. Vallee (Columbia University, New York, NY). Partial XKAP cDNA overlapping clones were isolated from a Xenopus oocyte cDNA library using as a probe a cDNA encoding SMAP (a gift from Pr Tokai, Osaka University Medical School, Osaka, Japan). Full-length XKAP cDNA was obtained by PCR on A6 cells cDNA and cloned into the pCR2.1 vector using the TA cloning kit (Invitrogen). Several constructs were prepared using internal restriction sites to express fragments of XKAP fused to either GST or GFP. For expression of GFP, dynamitin, and XKAP constructs, melanophores were plated on poly-lysinecoated coverslips and transfected with the FuGENE6 transfection reagent (Roche Diagnostics).
Melanosome tracking
To study the direction and length of melanosome movements, melanosomes were selected at random and observed for 60 s. Melanosomes exhibiting a linear movement of at least 2 µm with a velocity of at least 0.2 µm/s were counted. In addition, the direction of melanosome movement was determined, with movements away from the nucleus scored as anterograde and movements toward the nucleus scored as retrograde.
Biochemical techniques
Cells used in biochemical experiments were rinsed in PBS and detached from plates with a rubber policeman. Cells were lysed in IMB50 (50 mM imidizole, pH 7.4, 1 mM EGTA, 0.5 mM EDTA, 5 mM magnesium acetate, 175 mM sucrose, and 1 mM DTT) with protease inhibitors (PMSF, chymostatin, leupeptin, and pepstatin).
Immunoprecipitations were performed in IMB50. Cells were lysed and centrifuged at 16,000 g for 10 min. Extracts were precleared for 60 min with 10 µl normal rabbit IgG or myc antibody prebound to 100 µl of a 50% solution of protein A beads (Bio-Rad laboratories). Extracts were incubated for 60 min at RT with antibodies prebound to 25 µl of a 50% solution of protein A beads. Beads were washed in IMB50, and IMB50 with 250 mM NaCl. Antibodies used were as follows: normal rabbit IgG, anti-myc (Evan et al., 1985), K2.4 (monoclonal antikinesin II) (Cole et al., 1993), Kif3B (polyclonal antikinesin II) (a gift from Dr. Virgil Muresan, Case Western Reserve University, Cleveland, OH), 74.1 (monoclonal anti-DIC) (Steffen et al., 1997), polyclonal anti-DIC (Vaughan and Vallee, 1995), monoclonal anti-p150Glued (BD Transduction Laboratories), and polyclonal anti-p150Glued (Vaughan et al., 1999).
For peptide pull-down assays, extracts were incubated for 60 min at RT with 150 µl glutathione agarose beads (Sigma-Aldrich) to which protein was prebound. Beads were washed in IMB50 and IMB50 plus 200 mM NaCl. Beads were resuspended in laemmli buffer supplemented with 5 mM glutathione. These samples were probed with monoclonal anti-p150Glued and a polyclonal antibody against dynamitin (a gift from Dr. Erika Holzbaur, University of Pennsylvania, Philadelphia, PA).
Blot overlay assays were performed as described (Vaughan and Vallee, 1995) using recombinant p150Glued fragments 1811 and 600811. Overlays were probed with anti-p150Glued antibodies or anti-myc. For analysis of competition between kinesin II and dynein, recombinant DIC was probed by overlay using p150Glued 1811 alone or after preincubation with recombinant XKAP (1:1 or 1:2 molar ratios).
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Footnotes |
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Acknowledgments |
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Submitted: 11 October 2002
Revised: 13 December 2002
Accepted: 13 December 2002
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References |
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