Hestermann A and Gräf R (2004), The XMAP215-family protein DdCP224 is required ...

XB-ART-3446

BMC Cell Biol 2004 Jun 08;5:24. doi: 10.1186/1471-2121-5-24.

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The XMAP215-family protein DdCP224 is required for cortical interactions of microtubules.

Hestermann A , Gräf R .

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BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

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Species referenced: Xenopus
Genes referenced: actl6a ckap5 dnai1 nup62 sqstm1

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	Figure 1. Overexpression of DdCP224-ÎC-GFP causes a collapse of interphase microtubule arrays. (A, B, B') Confocal microscopy of GFP-Î±-tubulin cells (A; control) and DdCP224-ÎC-GFP cells showing brightest point projections of GFP fluorescence (A, B') or immunofluorescence staining (B) using the YL1/2 anti-tubulin antibody (Chemicon, Hofheim, Germany). Cells were fixed with methanol. DNA was stained with TOPRO3 (Molecular Probes, Hilversum, Netherlands) (blue). (C) Immunoblot of a cytosolic extract of DdCP224-ÎC-GFP cells stained with anti-DdCP-HindIII. This rabbit polyclonal antibody was raised against the recombinant His-tagged N-terminus of DdCP224 using the 5'-HindIII fragment of the DdCP224 coding sequence. As calculated by the ImageJ program, DdCP224-ÎC-GFP is overexpressed approximately 5-fold. Bar 2 Î¼m.
	Figure 2. Microtubule and centrosome dynamics upon overexpression of DdCP224-ÎC. GFP-Î±-tubulin control cells (A, see additional data file movie1.mov) and GFP-Î±-tubulin cells overexpressing DdCP224-ÎC (B, see additional data file movie2.mov) were analyzed by confocal 4D-microscopy as described [5]. Each image represents a brightest point z-projection of 5 confocal slices with a distance of 1 Î¼m each. The time is indicated in seconds. The movements of the centrosome shown in (C) for control cells and in (D) for DdCP224-ÎC/GFP-Î±-tubulin cells were calculated from 60 single images each using ImageJ.
	Figure 3. Cells with disrupted microtubule arrays show Golgi dispersal. Confocal microscopy of DdCP224-ÎC-GFP cells showing brightest point projections of immunofluorescence stainings using the Golgi-specific anti-comitin antibody [34] (A) and the YL1/2 anti-tubulin antibody (B). GFP fluorescence is shown in (C). The merged image (D) shows the Golgi in red, microtubules in green and GFP fluorescence in blue. Cells were fixed with formaldehyde/acetone. The two cells exhibiting Golgi dispersal and disrupted microtubule arrays are marked by an arrow. Bar 2 Î¼m.
	Figure 4. Cells with disrupted microtubule arrays show reduced cortical localization of dynein. Confocal microscopy of DdCP224-ÎC-GFP cells showing brightest point projections of immunofluorescence stainings using anti-dynein-Y7 [19] (A, B) and and the YL1/2 anti-tubulin antibody (A', B'). In both examples, the left cell exhibits a disrupted microtubule array and is characterized by reduced cortical distribution of the dynein heavy chain compared to the right cell which shows normal microtubules. Cells were fixed with formaldehyde/acetone. Bar 2 Î¼m.
	Figure 5. Coprecipitation of DdCP224 with dynein and DdEB1. Experiments were performed using cytosolic extracts from wildtype cells (strain AX2) (A-D), GFP-Ddp62 cells (E) and DdCP224-ÎC-GFP cells (F). The respective co-immunoprecipitation (Co-IP) experiment is given in bold letters on top of each subfigure. The respective antibodies used for immunoprecipitation and staining of the immunoblots are indicated. Abbreviations and antibodies: DHC, anti-dynein heavy chain Y7 [19]; DIC, anti-dynein intermediate chain [20]; DdCP, anti-DdCP224 mAb 2/165 [35] for immunoblot staining and anti-DdCP-HindIII for immunoprecipitation; DdEB1, anti-DdEB1 [16]; p62, anti-Ddp62; control, anti-rabbit or anti-rat (in case of DIC) preimmune serum.
	Figure 6. Colocalization of DdCP224, DdCP224-ÎC-GFP and dynein at the cell cortex. Confocal microscopy of DdCP224-ÎC-GFP cells showing brightest point projections of immunofluorescence stainings using anti-dynein-Y7 [19] (A) and anti-DdCP224 (2/165) (A', B'). GFP fluorescence is shown in (B). The merged images (A", B") shows endogenous DdCP224 in red and dynein (A) or DdCP-ÎC-GFP (B'), respectively, in green. Cells were fixed with formaldehyde/acetone. Bar 2 Î¼m.
	Figure 7. Model for the collapse of radial interphase microtubule arrays by disruption of cortical dynein/dynactin function. (A) Cortical dynein/dynactin in cooperation with DdCP224 provides the pulling force for maintenance of radial microtubule arrays. (B) Collapse of the radial microtubule array and altered centrosome positioning due to disruption of most cortical dynein/dynactin complexes and asymmetric pulling forces provided by only a few remaining functional cortical dynein/dynactin complexes (shown in red). This pathway may also involve further proteins which are not depicted in this model.

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