Dictyostelium EB1 is a genuine centrosomal component required for proper spindle formation

Abstract

EB1 proteins are ubiquitous microtubule-associated proteins involved in microtubule search and capture, regulation of microtubule dynamics, cell polarity, and chromosome stability. We have cloned a complete cDNA of Dictyostelium EB1 (DdEB1), the largest known EB1 homolog (57 kDa). Immunofluorescence analysis and expression of a green fluorescent protein-DdEB1 fusion protein revealed that DdEB1 localizes along microtubules, at microtubule tips, centrosomes, and protruding pseudopods. During mitosis, it was found at the spindle, spindle poles, and kinetochores. DdEB1 is the first EB1-homolog that is also a genuine centrosomal component, because it was localized at isolated centrosomes that are free of microtubules. Furthermore, centrosomal DdEB1 distribution was unaffected by nocodazole treatment. DdEB1 colocalized with DdCP224, the XMAP215 homolog, at microtubule tips, the centrosome, and kinetochores. Furthermore, both proteins were part of the same cytosolic protein complex, suggesting that they may act together in their functions. DdEB1 deletion mutants expressed as green fluorescent protein or maltose-binding fusion proteins indicated that microtubule binding requires homo-oligomerization, which is mediated by a coiled-coil domain. A DdEB1 null mutant was viable but retarded in prometaphase progression due to a defect in spindle formation. Because spindle elongation was normal, DdEB1 seems to be required for the initiation of the outgrowth of spindle microtubules.

Figure 1

Alignment of the DdEB1 and human EB1 amino acid sequences. Top trace (Dd), DdEB1; bottom trace (Hs), human EB1. Identical amino acids are shaded in black, similar ones in gray. The coiled-coil domain predicted with the Coilscan program (Lupas et al., 1991) is underlined and italicized in both sequences.

Figure 2

Cell cycle-dependent localization of DdEB1. DdEB1 (A) colocalizes with actin labeled with anti-Dictyostelium actin (Simpson et al., 1984) (A′) at protruding pseudopods. The merged image is displayed in A". The cell cycle stages shown are interphase (B), prophase (C), metaphase (D), and anaphase/telophase transition (E). Counterstaining was performed with the anti-DdCP224 monoclonal antibody 4/148 (B′, D′, and E′) (Gräf et al., 1999) or with the monoclonal anti-α-tubulin antibody YL1/2 (C′) (Chemicon, Hofheim, Germany). The arrows in D and D′ point to the kinetochore region, which is characterized by DdCP224 labeling. DNA (blue) was stained with TOPRO3. All images are maximum intensity projections of image stacks obtained by confocal microscopy and deconvolution, except A to A", and the insets in B and B′ show single confocal planes. The insets demonstrate the ring-like appearance of the interphase centrosome. Bar, 2 μm.

Figure 3

Centrosomal localization of DdEB1 does not require microtubules. (A) Treatment of wild-type cells with 30 μM nocodazole for 2 h did not reduce centrosomal labeling with DdEB1 antibodies. (B) DdEB1 is also present at isolated centrosomes. The centrosomes were counterstained with the anti-DdCP224 monoclonal antibody 4/148 (A′ and B′). DNA was stained with 4,6-diamidino-2-phenylindole (blue). Bar, 2 μm.

Figure 4

Microtubule binding of DdEB1 requires the presence of the coiled-coil domain. DdEB1 and its deletion mutants were expressed as GFP-fusion proteins. (A–D) GFP fluorescence; a scheme of the respective mutant is shown at the top of each panel. (A′–D′) Centrosomes were counterstained with the anti-DdCP224 monoclonal antibody 4/148. The cell shown in D is dinucleated. Such cells are not unusual in axenically grown Dictyostelium cultures and occur at the same frequency in the mutant as in wild-type cells. MTB, microtubule-binding domain; CC, coiled-coil. Bar, 2 μm.

Figure 5

Coiled-coil domain is required for homo-oligomerization. (A) Sizing of MBP-DdEB1 and its deletion mutants by native polyacrylamide gradient gel electrophoresis. (B) Sizing of MBP-DdEB1 by size exclusion chromatography. Numbers refer to the molecular masses in kilodaltons.

Figure 6

and Movie 1. DdEB1 interacts and colocalizes with DdCP224. (A–A") Colocalization of DdEB1 and DdCP224 at microtubule tips. Wild-type (AX2) Dictyostelium cells were stained with anti-DdEB1 antibodies (A) and the monoclonal anti-DdCP224 antibody 4/148 (A′). The inset in the merged image (A") is a magnification of the area indicated in the upper right part of the image. The images are maximum intensity projections of image stacks obtained by confocal microscopy and deconvolution. Movie 1 shows an animation of the deconvoluted confocal sections of the projection shown in A". (B) Coprecipitation of DdEB1 and DdCP224 from cytosolic Dictyostelium extracts from MBP-DdEB1 mutants or wild-type cells. Coprecipitates were loaded onto 12.5% acrylamide gels and blotted after electrophoresis. The matrix used for precipitation is given on the top and the antibodies used for Western blot staining are indicated on the bottom.

Figure 7

and Movie 2. Aberrant mitotic figures in DdEB1Δ mutants. The absence of DdEB1 in the knockout mutants (k/o) is shown in the Western blot (A), where cytosolic extracts of wild-type (wt) and DdEB1Δ mutants were probed with the anti-DdEB1 antibodies. The DdEB1Δ cell extract was prepared >2 mo after transformation. (B–D) Merged images of DdEB1Δ/GFP-α-tubulin mutants ∼2 wk after transformation of the DdEB1 knockout construct. The nuclei of a single cell are shown in each image. GFP fluorescence is shown in green, anti-DdCP224 staining for centrosomes in red, and DNA staining by TOPRO3 in blue. The images are maximum intensity projections of image stacks obtained by confocal microscopy and deconvolution. Bar, 2 μm. Movie 2 shows an animation of the deconvoluted confocal sections of the projection shown in D.

Figure 8

and Movie 3. DdEB1Δ mutants are retarded in prometaphase progression. Live cell observation of a dinucleated DdEB1Δ/GFP-α-tubulin cell by four-dimensional confocal microscopy. The time is indicated in seconds. Prometaphase lasts for >8 min before a spindle is formed and starts to elongate. The time-lapse movie is an animation of brightest point projections of z-stacks consisting of three confocal slices with a distance of 0.5 μm each. Z-stacks were recorded using 2 × 2 binning, a time delay of 2.5 s between each stack and an exposure 500 ms/frame.