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First published online December 31, 2003
doi: 10.1242/10.1242/jcs.00869

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Maternally expressed and partially redundant ß-tubulins in Caenorhabditis elegans are autoregulated

Gregory C. Ellis^1,*, Jennifer B. Phillips¹, Sean O'Rourke¹, Rebecca Lyczak^1, and Bruce Bowerman^1,

¹ Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA

View larger version (55K): [in a new window]   Fig. 2. Microtubule-dependent processes are defective in tbb-2 mutant embryos. (A) Nomarski differential interference contrast (DIC) micrographs of wild-type embryos beginning after the completion of meiosis II and ending during the first cytokinesis. In all panels in this and other figures, anterior is to the left and posterior to the right. m, maternal pronucleus; p, paternal pronucleus. Arrowheads mark positioning of the centrosomes during centrosome/pronuclear rotation and during anaphase (Movies 1-4, http://jcs.biologists.org/supplemental). (B) Centration in wild-type and tbb-2 mutant embryos. Each X represents the position along the long axis of the midpoint between the two centrosomes of the centrosome/pronuclear complex at nuclear envelope breakdown. If the pronuclei did not meet, the measurement was taken during nuclear envelope breakdown of the centrosome/paternal pronuclear complex. (C) Extent of centrosome/pronuclear complex rotation in wild type and in tbb-2 mutants, showing the angle of the mitotic spindle relative to the long axis just after nuclear envelope breakdown. (D) Anaphase spindle position in wild-type and in tbb-2 mutant embryos. Each bar represents the spindle position roughly 2 minutes after nuclear envelope breakdown in one embryo. Percentage of embryo length indicates the average spindle position relative to the anterior pole of the embryo.

View larger version (13K): [in a new window]   Fig. 1. Structure and homology of C. elegans ß-tubulin. (A) ß-tubulin domains indicated as described by savage et al. (Savage et al., 1994). or362 is a glycine to glutamic acid substitution in a GTP-binding domain, while t1623 is a valine to methionine substitution in an assembly domain. (B) The exon/intron structure of tbb-2 and approximate location of the deletion gk129.

View larger version (68K): [in a new window]   Fig. 3. Spindle microtubules and TBB-2 protein in wild-type and tbb-2 mutant embryos and extracts. (A) Western blot showing TBB-2 levels relative to an actin loading control, in embryo extracts from wild-type, gk129 and or362 animals (see Materials and Methods). or362 embryo extracts were prepared from worms matured at permissive or restrictive temperatures. (B) Western blot showing TBB-2 levels relative to an actin loading control in whole worm extracts prepared from wild-type and t1623 animals. (C) Indirect immunofluorescence images of wild-type and tbb-2 mutant embryos stained with antibodies that recognize -tubulin (red) and TBB-2 (green); DNA was stained with TOTO (blue).

View larger version (69K): [in a new window]   Fig. 4. TBB-1 is upregulated in the absence of TBB-2. (A) Western blots showing TBB-1 levels in wild-type and tbb-2 embryo extracts that have reduced levels of TBB-2. Actin was used as an internal loading control. (B) Western blots showing TBB-1 levels in wild-type and tbb-2 (t1623) whole worm extracts, which have wild-type levels of TBB-2. (C) Fixed wild-type and mutant embryos stained with antibodies that recognize TBB-1 (green); DNA was stained with TOTO (blue). (D) Indirect immunofluorescence image of a fixed tbb-1(3' UTR RNAi) embryo stained with antibodies that recognize -tubulin (red) and TBB-1 (green); DNA was stained with TOTO (blue).