- Research article
- Open Access
The XMAP215-family protein DdCP224 is required for cortical interactions of microtubules
© Hestermann and Gräf; licensee BioMed Central Ltd. 2004
- Received: 20 March 2004
- Accepted: 08 June 2004
- Published: 08 June 2004
Interactions of peripheral microtubule tips with the cell cortex are of crucial importance for nuclear migration, spindle orientation, centrosome positioning and directional cell movement. Microtubule plus end binding proteins are thought to mediate interactions of microtubule tips with cortical actin and membrane proteins in a dynein-dependent manner. XMAP215-family proteins are main regulators of microtubule plus end dynamics but so far they have not been implicated in the interactions of microtubule tips with the cell cortex.
Here we show that overexpression of an N-terminal fragment of DdCP224, the Dictyostelium XMAP215 homologue, caused a collapse of the radial microtubule cytoskeleton, whereby microtubules lost contact with the cell cortex and were dragged behind like a comet tail of an unusually motile centrosome. This phenotype was indistinguishable from mutants overexpressing fragments of the dynein heavy chain or intermediate chain. Moreover, it was accompanied by dispersal of the Golgi apparatus and reduced cortical localization of the dynein heavy chain indicating a disrupted dynein/dynactin interaction. The interference of DdCP224 with cortical dynein function is strongly supported by the observations that DdCP224 and its N-terminal fragment colocalize with dynein and coimmunoprecipitate with dynein and dynactin.
Our data show that XMAP215-like proteins are required for the interaction of microtubule plus ends with the cell cortex in interphase cells and strongly suggest that this function is mediated by dynein.
- Cell Cortex
- Cleavage Furrow
- Microtubule Array
- Spindle Orientation
- Dictyostelium Cell
Interactions of peripheral microtubule plus ends with the cell cortex are of crucial importance for nuclear migration, spindle orientation, centrosome positioning and directional cell movement. Cortical dynein and dynactin components play an important role in mediating such interactions, in cooperation with a microtubule plus end complex consisting of a growing number of microtubule-associated proteins [1, 2]. Only little is known about a role of the XMAP215-family (named after their Xenopus representative) of microtubule-associated proteins in this process. The ubiquitous occurrence of these proteins in all kinds of organisms including plants suggests general and indispensable functions . In addition to their role as promoters of microtubule elongation, further functions in microtubule growth and nucleation [4, 5] and centrosome duplication [5, 6] have been described. In most species, XMAP215-family proteins are elongated, monomeric molecules with a size of approximately 230 kDa . By contrast, the yeast homologues (Stu2p in S. cerevisiae, dis1 and Alp14 in S. pombe) occur as dimers and are less than half as long as their counterparts in Dictyostelium and higher cells [8–10]. In budding yeast the major function of Stu2p is observed during mitosis where it regulates microtubule dynamics and is required for chromosome segregation [11–13]. Furthermore, Stu2p interacts with the cortical protein Kar9p  and genetic evidence, i.e. crossings of temperature sensitive stu2p mutants with kar9Δ or dynein (dhc1Δ) mutants, suggests that Stu2p plays a role in the Kar9p dependent pathway for spindle orientation . However, until this work there was no evidence for a physical interaction of the long, monomeric members of the XMAP215-family with dynein or a Kar9p-like protein such as APC , and there were no data supporting a role in microtubule plus-end/cell cortex interactions in interphase cells.
Like XMAP215, its Dictyostelium homologue, DdCP224, is both a microtubule-associated protein and a genuine centrosomal component [6, 15]. Furthermore, it was the first member of the XMAP215-family that was clearly localized at microtubule plus ends, both at kinetochores and microtubule tips near the cell cortex [6, 16]. Overexpression of the N-terminal half of DdCP224 as a GFP-fusion protein caused a cytokinesis defect . Since cleavage furrow positioning is determined by the pattern of interaction of astral microtubules with the cell cortex , both the cytokinesis defect of ΔC-GFP overexpressing mutants and the detection of DdCP224 at microtubule tips were in agreement with a novel role of DdCP224 in the crosstalk of microtubule tips with the cell cortex. Here we provide evidence for such a function of XMAP215-like proteins and suggest that it is mediated through the interaction with dynein.
To elucidate how overexpression of the DdCP224-ΔC-GFP fragment could cause the mutant phenotype, we investigated its intermolecular interactions. Since the DdCP224-ΔC-GFP fragment did not coimmunoprecipitate with either dynein or DdEB1 (data not shown), we wondered whether it might interact with dynactin. Due to the lack of specific antibodies against dynactin components, we cloned the Dictyostelium homologue of dynactin-p62 (Ddp62) as a marker for dynactin and raised antibodies against the recombinant protein. These antibodies showed only weak staining of denatured Ddp62 in Western blots. However, they were capable of specific immunoprecipitation of a GFP-Ddp62 fusion protein from cytosolic extracts of Dictyostelium GFP-Ddp62 mutants (Fig. 5E). Hence, we concluded that these antibodies showed a higher avidity to native than to denatured Ddp62. Using these antibodies we could demonstrate by co-immunoprecipitation that Ddp62 binds to endogenous DdCP224 and to the cytosolic DdCP224-ΔC-GFP fragment (Fig. 5F).
What could be the role of the cortical and the microtubule tip populations of DdCP224  in microtubule/cortex interactions? At microtubule tips it could play a role in the capturing of microtubule plus ends at cortical sites, however, since DdCP224, like XMAP215, promotes microtubule growth , it is likely that the major function of tip-localized DdCP224 is the regulation of microtubule plus-end dynamics and the prevention of catastrophes induced by antagonistic Kin I-family kinesins . By contrast, the cortical population of DdCP224 that colocalizes with dynein appears to be required for proper dynein function at the cortex. This is supported by two observations. First, DdCP224 binds to dynein/dynactin and second, overexpression of the DdCP224-ΔC-fragment disrupts cortical dynein function. Excess amounts of this DdCP224 fragment appear to interfere with the interaction between dynein and dynactin, since the characteristic collapse of the radial microtubule array was accompanied by reduced cortical dynein localization and by Golgi dispersal which is indicative for disrupted dynein/dynactin interaction . The simplest explanation is that the DdCP224-ΔC-fragment sequesters Ddp62 and possibly other dynactin components in the cytosol which are then missing at the cell cortex where they are required for proper dynein function. The cytokinesis defect observed upon overexpression of the DdCP224-ΔC-GFP fragment  also agrees with an active role of DdCP224 in the interaction of microtubule tips with cortical sites, since these interactions are involved in cleavage furrow positioning .
The pathway of dynein/dynactin/DdCP224-dependent cortical interactions of interphase microtubules reported herein has to be distinguished from that of spindle orientation in mitotic yeast cells. In the latter case, Stu2p is involved in Kar9p-dependent capture of cytoplasmic MT plus-ends at the bud tip [12, 13], a process that essentially requires yeast EB1 (Bim1p) at the MT tips [28, 29]. Although Dictyostelium EB1 interacts with both DdCP224 and dynein, the process of MT/cortex interaction described here is clearly independent of DdEB1. DdEB1 null mutants showed only a defect in mitotic spindle formation, but neither a defect in microtubule organization or centrosome positioning, nor a cytokinesis defect .
Taken together, our results demonstrate for the first time that XMAP215-family proteins such as DdCP224 are involved in microtubule plus-end/cell cortex interactions and centrosome positioning in interphase cells and that this is mediated through an interaction of DdCP224 with dynein and dynactin.
Generation of the GFP-α-tubulin/DdCP224-ΔC mutant
The Dictyostelium vector for expression of the untagged N-terminal 813 amino acids of DdCP224 was constructed by deletion of the GFP sequence in pΔC-GFP . It was then transformed into a Dictyostelium cell line expressing GFP-α-tubulin . Cells were cultured as described earlier .
Cloning of Ddp62, protein expression and generation of polyclonal antibodies
The gene encoding the Dictyostelium homologue of the p62 subunit of dynactin (Ddp62; DictybaseID DDB0206421) was identified in the Dictyostelium genome project . Its complete coding sequence (1647 bp) was amplified by PCR using an oligo dT-primed cDNA library  as a template. The Ddp62 cDNA was re-amplified using either BamHI and PstI linker primers for cloning into the pMALc2 (NEB, Frankfurt, Germany) or SalI and BamHI linker primers for cloning into pIS77, a vector obtained after replacement of the discoidin promoter of pDiscGFPSSEB2  by the actin6 promoter. The former construct was used for protein expression in E. coli, the latter for expression of a GFP-Ddp62 fusion protein in Dictyostelium. The MBP-Ddp62 fusion protein expressed in E. coli was purified by affinity chromatography on amylose resin and used for custom immunization of two rabbits (Pineda Antikörperservice, Berlin, Germany). Both antisera showed the same characteristics.
Immunoprecipitation was performed essentially as described previously . In brief, 2 × 108 cells (80 ml) were lysed in 5 ml of lysis buffer (50 mM Hepes, 100 mM NaCl, 4 mM EGTA, 2 mM MgCl2, 10% sucrose, 0.3% NP40, 1 × protease inhibitor cocktail ). A cytosolic extract was obtained after centrifugation at 14.000 × g for 10 min at 4°C. After incubation of 0.6 ml of cytosolic extract with 10 μg of purified antibodies or 1.5 μl of antiserum for 1 h at 4°C, 20 μl of Protein G beads (50% slurry preincubated with 0.1% BSA in Tris-buffered saline) were added for a further incubation for 1 h at 4°C in a rotator. Beads were washed 4 times with lysis buffer, resuspended with 30 μl of SDS sample buffer (10% SDS, 125 mM Tris/HCl, pH = 6.8, 50 mM DTT, 5% glycerol) and subjected to electrophoresis and Western blotting as described previously .
Microscopy and image processing
Immunofluorescence microscopy and live cell observation was performed as described previously [5, 33]. All microscopic images were acquired on a Zeiss Axiovert 200M/510META confocal microscope equipped with a 63x/1.4N.A. lens.
We are very grateful to Mike Koonce, Rex Chisholm and Michael Schleicher for providing antibodies against the dynein heavy chain, dynein intermediate chain and comitin, respectively. We also would like to thank Manfred Schliwa for his continuous support, Markus Rehberg and Alexandra Lepier for critical comments and Thi-Hieu Ho for expert technical assistance. Supported by the DFG (SFB413 and GR1642/2-1).
- Schuyler SC, Pellman D: Microtubule "plus-end-tracking proteins": The end is just the beginning. Cell. 2001, 105: 421-424. 10.1016/S0092-8674(01)00364-6.View ArticlePubMedGoogle Scholar
- Dujardin DL, Vallee RB: Dynein at the cortex. Curr Opin Cell Biol. 2002, 14: 44-49. 10.1016/S0955-0674(01)00292-7.View ArticlePubMedGoogle Scholar
- Ohkura H, Garcia MA, Toda T: Dis1/TOG universal microtubule adaptors - one MAP for all?. J Cell Sci. 2001, 114: 3805-3812.PubMedGoogle Scholar
- Popov A, Severin F, Karsenti E: XMAP215 Is Required for the Microtubule-Nucleating Activity of Centrosomes. Curr Biol. 2002, 12: 1326-10.1016/S0960-9822(02)01033-3.View ArticlePubMedGoogle Scholar
- Gräf R, Euteneuer U, Ho TH, Rehberg M: Regulated Expression of the Centrosomal Protein DdCP224 Affects Microtubule Dynamics and Reveals Mechanisms for the Control of Supernumerary Centrosome Number. Mol Biol Cell. 2003, 14: 4067-4074. 10.1091/mbc.E03-04-0242.PubMed CentralView ArticlePubMedGoogle Scholar
- Gräf R, Daunderer C, Schliwa M: Dictyostelium DdCP224 is a microtubule-associated protein and a permanent centrosomal resident involved in centrosome duplication. J Cell Sci. 2000, 113: 1747-1758.PubMedGoogle Scholar
- Cassimeris L, Gard D, Tran PT, Erickson HP: XMAP215 is a long thin molecule that does not increase microtubule stiffness. J Cell Sci. 2001, 114: 3025-3033.PubMedGoogle Scholar
- Wang PJ, Huffaker TC: Stu2p: A microtubule-binding protein that is an essential component of the yeast spindle pole body. J Cell Biol. 1997, 139: 1271-1280. 10.1083/jcb.139.5.1271.PubMed CentralView ArticlePubMedGoogle Scholar
- Nabeshima K, Kurooka H, Takeuchi M, Kinoshita K, Nakaseko Y, Yanagida M: p93dis1, which is required for sister chromatid separation, is a novel microtubule and spindle pole body-associating protein phosphorylated at the Cdc2 target sites. Genes Dev. 1995, 9: 1572-1585.View ArticlePubMedGoogle Scholar
- Garcia MA, Vardy L, Koonrugsa N, Toda T: Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint. EMBO J. 2001, 20: 3389-3401. 10.1093/emboj/20.13.3389.PubMed CentralView ArticlePubMedGoogle Scholar
- Van Breugel M, Drechsel D, Hyman A: Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer. J Cell Biol. 2003, 161: 359-369. 10.1083/jcb.200211097.PubMed CentralView ArticlePubMedGoogle Scholar
- Kosco KA, Pearson CG, Maddox PS, Wang PJ, Adams IR, Salmon ED, Bloom K, Huffaker TC: Control of microtubule dynamics by stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast. Mol Biol Cell. 2001, 12: 2870-2880.PubMed CentralView ArticlePubMedGoogle Scholar
- Miller RK, Cheng SC, Rose MD: Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules. Mol Biol Cell. 2000, 11: 2949-2959.PubMed CentralView ArticlePubMedGoogle Scholar
- Bloom K: It's a kar9ochore to capture microtubules. Nat Cell Biol. 2000, 2: E96-8. 10.1038/35014089.View ArticlePubMedGoogle Scholar
- Popov AV, Pozniakovsky A, Arnal I, Antony C, Ashford AJ, Kinoshita K, Tournebize R, Hyman AA, Karsenti E: XMAP215 regulates microtubule dynamics through two distinct domains. EMBO J. 2001, 20: 397-410. 10.1093/emboj/20.3.397.PubMed CentralView ArticlePubMedGoogle Scholar
- Rehberg M, Gräf R: Dictyostelium EB1 Is a Genuine Centrosomal Component Required for Proper Spindle Formation. Mol Biol Cell. 2002, 13: 2301-2310. 10.1091/mbc.E02-01-0054.PubMed CentralView ArticlePubMedGoogle Scholar
- Neujahr R, Albrecht R, Kohler J, Matzner M, Schwartz JM, Westphal M, Gerisch G: Microtubule-mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells. J Cell Sci. 1998, 111: 1227-1240.PubMedGoogle Scholar
- Koonce MP, Kohler J, Neujahr R, Schwartz JM, Tikhonenko I, Gerisch G: Dynein motor regulation stabilizes interphase microtubule arrays and determines centrosome position. EMBO J. 1999, 18: 6786-6792. 10.1093/emboj/18.23.6786.PubMed CentralView ArticlePubMedGoogle Scholar
- Koonce MP, Samso M: Overexpression of cytoplasmic dynein's globular head causes a collapse of the interphase microtubule network in Dictyostelium. Mol Biol Cell. 1996, 7: 935-948.PubMed CentralView ArticlePubMedGoogle Scholar
- Ma S, Trivinos Lagos L, Gräf R, Chisholm RL: Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium. J Cell Biol. 1999, 147: 1261-1274. 10.1083/jcb.147.6.1261.PubMed CentralView ArticlePubMedGoogle Scholar
- Koonce MP, Khodjakov A: Dynamic microtubules in Dictyostelium. J Muscle Res Cell Motil. 2002, 23: 613-619. 10.1023/A:1024446821701.View ArticlePubMedGoogle Scholar
- Dujardin DL, Barnhart LE, Stehman SA, Gomes ER, Gundersen GG, Vallee RB: A role for cytoplasmic dynein and LIS1 in directed cell movement. J Cell Biol. 2003, 163: 1205-1211. 10.1083/jcb.200310097.PubMed CentralView ArticlePubMedGoogle Scholar
- Schliwa M, Euteneuer U, Gräf R, Ueda M: Centrosomes, microtubules and cell migration. Biochem Soc Symp. 1999, 65: 223-231.PubMedGoogle Scholar
- Palazzo AF, Joseph HL, Chen YJ, Dujardin DL, Alberts AS, Pfister KK, Vallee RB, Gundersen GG: Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr Biol. 2001, 11: 1536-1541. 10.1016/S0960-9822(01)00475-4.View ArticlePubMedGoogle Scholar
- Etienne-Manneville S, Hall A: Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell. 2001, 106: 489-498. 10.1016/S0092-8674(01)00471-8.View ArticlePubMedGoogle Scholar
- Kinoshita K, Habermann B, Hyman AA: XMAP215: a key component of the dynamic microtubule cytoskeleton. Trends Cell Biol. 2002, 12: 267-273. 10.1016/S0962-8924(02)02295-X.View ArticlePubMedGoogle Scholar
- Burkhardt JK, Echeverri CJ, Nilsson T, Vallee RB: Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. J Cell Biol. 1997, 139: 469-484. 10.1083/jcb.139.2.469.PubMed CentralView ArticlePubMedGoogle Scholar
- Maekawa H, Usui T, Knop M, Schiebel E: Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions. Embo J. 2003, 22: 438-449. 10.1093/emboj/cdg063.PubMed CentralView ArticlePubMedGoogle Scholar
- Hwang E, Kusch J, Barral Y, Huffaker TC: Spindle orientation in Saccharomyces cerevisiae depends on the transport of microtubule ends along polarized actin cables. J Cell Biol. 2003, 161: 483-488. 10.1083/jcb.200302030.PubMed CentralView ArticlePubMedGoogle Scholar
- Eichinger L, Noegel AA: Crawling into a new era-the Dictyostelium genome project. Embo J. 2003, 22: 1941-1946. 10.1093/emboj/cdg214.PubMed CentralView ArticlePubMedGoogle Scholar
- Daunderer C, Gräf R: Molecular analysis of the cytosolic Dictyostelium gamma-tubulin complex. Eur J Cell Biol. 2002, 81: 175-184.View ArticlePubMedGoogle Scholar
- Harlow E, Lane D: Using Antibodies - A Laboratory Manual. 1999, Cold Spring Harbour, New York, Cold Spring Harbour Laboratory PressGoogle Scholar
- Gräf R, Euteneuer U, Ueda M, Schliwa M: Isolation of nucleation-competent centrosomes from Dictyostelium discoideum. Eur J Cell Biol. 1998, 76: 167-175.View ArticlePubMedGoogle Scholar
- Weiner OH, Murphy J, Griffiths G, Schleicher M, Noegel AA: The actin-binding protein comitin (p24) is a component of the golgi apparatus. J Cell Biol. 1993, 123: 23-34. 10.1083/jcb.123.1.23.View ArticlePubMedGoogle Scholar
- Gräf R, Daunderer C, Schliwa M: Cell cycle-dependent localization of monoclonal antibodies raised against isolated Dictyostelium centrosomes. Biol Cell. 1999, 91: 471-477. 10.1016/S0248-4900(99)80088-4.View ArticlePubMedGoogle Scholar
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