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First published online 17 July 2006
doi: 10.1242/jcs.03039

Journal of Cell Science 119, 3238-3248 (2006)
Published by The Company of Biologists 2006
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{alpha}4-Tubulin is involved in rapid formation of long microtubules to push apart the daughter centrosomes during earlyx Drosophila embryogenesis

Zsolt Venkei1, Imre Gáspár1, Gábor Tóth2 and János Szabad1,*

1 Maternal Effect and Embryogenesis Research Group of the Hungarian Academy of Sciences at the University of Szeged, Faculty of Medicine, Department of Biology, Somogyi B. u. 4, H-6720 Szeged, Hungary
2 University of Szeged, Faculty of Medicine, Department of Medical Chemistry, Szeged, Hungary


Figure 1
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  		Fig. 1. Time-lapse series of optical sections throughout the twelfth cleavage cycle following injection of egg cytoplasm into embryos. Microtubules are highlighted by {alpha}1-tubulin-GFP. The embryos were injected during anaphase of the eleventh cleavage cycle. The injected cytoplasm samples were taken from wild-type eggs (Wild type) or eggs from Kavar18c/- females (Kavar18c/-). E82K-{alpha}4-tubulin, introduced in the Kavar18c/- egg cytoplasm, slowed down the separation of the daughter centrosomes (white arrows). The partially separated centrosomes either organize the formation of short microtubules that never assemble to a spindle apparatus (middle column of panels), or fail to localize the nuclei properly (right column of panels; adjoining nuclei in the boxed area); as a consequence tripolar spindles form. Asterisks indicate sites of cytoplasm injection; time after injection is shown in the first column of panels but applies to the entire row; arrows indicate centrosomes, open arrows indicate interpolar microtubule bundles.

 

Figure 2
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  		Fig. 2. Specificity of the monoclonal DM1A antibody to {alpha}1-tubulin and {alpha}3-tubulin, the constitutively expressed and evolutionary highly conserved {alpha}-tubulin isoforms, and of rabbit anti-{alpha}4-tubulin polyclonal antibody that recognizes only {alpha}4-tubulin. Notice that DM1A does not recognize {alpha}4-tubulin. There is no {alpha}4-tubulin in egg cytoplasm of kavar0/- females. (Dilutions were 500-fold for DM1A and 2000-fold for anti-{alpha}4-tubulin.)

 

Figure 3
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  		Fig. 3. Localization of {alpha}1-tubulin and {alpha}3-tubulin as well as {alpha}4-tubulin throughout the eleventh cleavage cycle in a wild-type embryo. The constitutively expressed {alpha}1-tubulin and {alpha}3-tubulin isoforms were detected with the monoclonal DM1A primary and a Texas-Red-labeled fluorescent secondary antibody (merged images). {alpha}4-tubulin was detected by an affinity-purified rabbit polyclonal anti-{alpha}4-tubulin primary antibody and an FITC-labeled fluorescent secondary antibody (merged images). DNA appears in blue (merged images). Arrowheads indicate interpolar microtubules.

 

Figure 4
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  		Fig. 4. The maternal {alpha}4-tubulin isoform is enriched in the interpolar microtubulues. The {alpha}1-tubulin and {alpha}3-tubulin and also the {alpha}4-tubulin isoforms were stained in fixed cleavage embryos as described in Fig. 3 ({alpha}1-tubulin and {alpha}3-tubulin, red; {alpha}4-tubulin, green, DNA, blue) and analyzed in optical sections prepared in an Olympus FV1000 confocal microscope. To reveal the concentrations of {alpha}1-tubulin and {alpha}3-tubulin as well as {alpha}4-tubulin over the nuclear region, we set one line across the centrosomes and another line crossing the interpolar microtubule region. An arc was set to follow the region of the interpolar microtubules. We then determined the distribution of signal intensities (ranging from 0 to 255 arbitrary units) in the pixels along the two lines and the arc. The pixel intensities reflect fluorescence intensities and the amounts of the different types of tubulins. The curves over the lines illustrate - as examples - intensity distributions for a single nuclear region. The intensity distributions along the arc appear above the straight lines below the images. Altogether, 20 nuclear regions were analyzed: five nuclei of four embryos each. The peak intensities (average ± s.d.; n=40) over the centrosome regions and at the intersections in the interpolar microtubules (along the arches) are shown in the figure. The analysis revealed highly significant accumulation of {alpha}4-tubulin in the interpolar microtubules (P<0.01, t-test). Merged image shows {alpha}4-tubulin, green; {alpha}1-tubulin and {alpha}3-tubulin, red; DNA, blue.

 

Figure 5
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  		Fig. 5. Localization of {alpha}1-tubulin and {alpha}3-tubulin (red), {alpha}4-tubulin (green) and DNA (blue) in the first mitotic spindle in wild-type embryos, Kavar18c/- and kavar0/- females. The first and only monopolar spindle that appears in eggs of the mutant females is a tassel of short microtubules.

 

Figure 6
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  		Fig. 6. E82K-{alpha}4-tubulin is incorporated into the microtubules during in vitro tubulin-polymerization. Microtubule formation was induced in egg extracts of +/- (contains one wild-type {alpha}Tub67C gene) and Kavar18c/- females. Formed microtubules were pelleted. Egg extracts, supernatant and microtubule-containing pellet were analyzed by SDS PAGE stained with Coomassie Blue and western blot. The {alpha}1-tubulin and the {alpha}3-tubulin isoforms were detected with monoclonal DM1A primary and a Texas-Red-labeled fluorescent secondary antibody, {alpha}4-tubulin by a polyclonal anti-{alpha}4-tubulin primary and an FITC-labeled fluorescent secondary antibody.

 

Figure 7
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  		Fig. 7. Kinetics of in vitro tubulin-polymerization. Length distribution of the forming microtubules plotted against the time allowed for tubulin polymerization. White and grey arrows and bars correspond to egg extracts of females with one wild-type {alpha}Tub67C gene (+/-, control) and egg extracts of females not carrying functional {alpha}Tub67C genes (kavar0/-). The wide arrows represent preparations in which only nucleation seeds appeared. The five horizontal lines in the bars correspond to 10th, 25th, 50th, 75th and 90th percentiles, respectively (as shown on the right). For details of tubulin polymerization see supplementary material Fig. S1. White and gray diamonds represent average microtubule lengths for control and null-mutant, respectively. Curves (double line, control; single line, null) were fitted to these points.

 

Figure 8
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  		Fig. 8. Ectopically expressed {alpha}4-tubulin and E82K-{alpha}4-tubulin are incorporated into the microtubules in the neuroblast as shown by the encircled spindle apparatus. Ectopic expression of {alpha}4-tubulin and E82K-{alpha}4-tubulin was achieved by driving UAS-{alpha}Tub67C and UAS-Kavar18c transgenes with the nervous-system-specific elav-Gal4 driver. {alpha}1-tubulin and {alpha}3-tubulin (red) were detected with monoclonal DM1A primary and a Texas-Red-labeled fluorescent secondary antibody, {alpha}4-tubulin and E82K-{alpha}4-tubulin (both green) with a polyclonal primary anti-{alpha}4-tubulin and a FITC-labeled fluorescent secondary antibody; DNA is stained blue.

 

Figure 9
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  		Fig. 9. Diameter of late interphase cleavage nuclei throughout the cleavage cycles in Drosophila melanogaster wild-type embryos. The distance between opposite poles was calculated assuming ball-shaped nuclei.

 

Figure 10
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  		Fig. 10. Simplified model to explain the mechanism of daughter centrosome separation and migration in the early cleavage (cycles 2-10) Drosophila embryos. The interpolar microtubules, nucleated by the daughter centrosomes (large arrowheads) exert pushing force and begin to separate the daughter centrosomes. Microtubules containing {alpha}4-tubulin bend around the nuclear envelope while they grow. Dyneins fixed to the nuclear envelope (not shown) keep the microtubules attached and use them as a migration route to pull the centrosomes in the indicated directions. Centrosome migration stops when the interpolar microtubules bend away from the nuclear envelope and the centrosomes organize symmetric array of microtubules equalizing inward and outward forces.

 

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© The Company of Biologists Ltd 2006