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First published online November 9, 2005
doi: 10.1242/10.1242/jcs.02652

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Tum/RacGAP50C provides a critical link between anaphase microtubules and the assembly of the contractile ring in Drosophila melanogaster

¹ ARC Special Research Centre for the Molecular Genetics of Development and Molecular Genetics and Evolution Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia
² Department of Biology, Duke University, Durham, NC 27705-1000, USA

View larger version (83K): [in a new window]   Fig. 1. Cytokinesis fails in the embryonic epithelium of tum mutant embryos. (A) Diagram of the Tum protein showing the location of the two premature stop codon mutations in the tumDH15 and tumAR2 alleles, the Pbl- and Pav-interacting regions deleted in the tumPav and tumPbl deletions, and the amino acids deleted in the tumEIE and tumYRL deletions. Numbers indicate the amino acids deleted in the transgenes or altered to stop codons in the mutants. (B-D) Binucleate cells accumulate in the tumDH15 mutant epithelium. DNA (B, and blue in D) and F-actin (C and green in D). Examples of binucleate cells in a stage 11 mutant embryo are indicated with asterisks in D. (E-G) Expression of UAS-tum rescues the cytokinetic defect of tumDH15 embryos. DNA (E and blue in G), Tum (F and red in G) and F-actin (green in G). Tum was expressed in mutant embryos using the prdGal4 driver. The bracket below panels F and G indicates the approximate position of the prd stripe. Tum is located in midbody remnants and at the cytokinetic furrow of a telophase cell (F and G, arrow). Binucleate cells are abundant in non-expressing stripes of cells (some marked by asterisks in G). Bar, 5 µm.

View larger version (86K): [in a new window]   Fig. 2. Anaphase microtubule reorganization fails in tum mutant cells. (A-D) Tum localisation in wild-type dividing cells. DNA (A and blue in D), Tum (B and red in D) and microtubules (C and green in D). Prometaphase cells show diffuse Tum staining (asterisks in B). An anaphase cell (arrowhead, B) has Tum localised at the cortex and in microtubule bundles. A telophase cell (arrow, B) has partially completed cytokinesis and has a ring of Tum. Residual midbodies mark the positions of previous divisions (arrows, D). This is a merged Z-stack representing a total 4-µm-thick slice. (E-H) Tum localisation in dividing cells of the tumDH15 mutant at stage 12. Tum alone (F) and merged (H, in red) with microtubules in green and DNA in blue. Tum is not detectable in embryos at this time (F). Metaphase cells (arrows, G) appear normal, whereas anaphase and telophase cells (asterisks, G) have a variety of aberrant microtubule bundles. This is a merged Z-stack representing a total 1-µm-thick slice. Bar, 5 µm.

View larger version (64K): [in a new window]   Fig. 3. Loss of Tum results in mislocalisation of other cytokinetic furrow components in mutant embryos. (A,B) The localisation of AurB (green) is shown alone in the upper panels, and merged with the microtubule (red) and DNA (blue) images in the lower panels in wild-type embryos (A) and tumDH15 embryos (B). AurB is associated with the chromosomes of metaphase cells (arrowheads, A and B) in mutant and wild-type embryos. At anaphase AurB moves to the central spindle and cortex in wild-type cells (arrow, A) but is not localised correctly in mutant cells (arrow, B). (C,D) The localisation of Diaphanous (green) is shown alone in the upper panels, and merged with the microtubule (red) and DNA (blue) images in the lower panels in wild-type (C) and tumDH15 (D) embryos. Diaphanous is cortical throughout the cell cycle, but concentrates at the midzone cortex of wild-type telophase cells (arrowheads, C). In tumDH15 mutants, Diaphanous does not concentrate in the midzone cortex (arrowhead, D). (E,F,G) The localisation of Pbl-GFP (green) is shown alone in the upper panels and merged with the microtubule (red E, G), Tum (red, F) and DNA (blue) images in the lower panels in wild-type (E,F) and tumDH15 (G) embryos. At anaphase, Pbl-GFP is concentrated in the midzone cortex in normal cells (arrowheads, E,F) but remains cortical throughout mitosis in tumDH15 mutant cells (arrowheads, G). (H,I) The localisation of Anillin (green) is shown alone in the upper panels, and merged with Tum (red) and DNA (blue) in the lower panels in wild-type (H) and tumDH15 (I) embryos. Anillin concentrates at the midzone at anaphase (arrowhead, H) and concentrates in the furrow as cells undergo cytokinesis (arrow, H). In tum mutant cells, Anillin is cortical in metaphase (asterisk, I) and does not concentrate in the midzone cortex during telophase (arrow, I). (J,K) The localisation of Pav-KLP (green) is shown alone in the upper panels, and merged with the microtubule (red) and DNA (blue) images in the lower panels in wild type (J) and tumDH15 (K) embryos. In wild-type embryos, Pav-KLP concentrates at the furrow at anaphase (arrow, J) and becomes concentrated in the midbody as cytokinesis progresses (arrowhead, J). In tum mutant embryos, no Pav-KLP is detected in anaphase or telophase cells (arrowhead, K). Bar, 5 µm. Grayscale images for all color channels can be viewed in supplementary material Fig. S3.

View larger version (74K): [in a new window]   Fig. 4. Elevated Pav-KLP protein levels occur in stripes of cells expressing all but the UAS-tumPav transgene. In these experiments transgene-derived Tum localisation was detected with a myc antibody (shown in green), and Pav-KLP is shown in red. (A-C) The arrowhead in C indicates two anaphase cells with Pav-KLP and Tum colocalising at the furrow sites in a prd-GAL4-induced expression stripe. Midbody remnants containing both proteins are also evident. In panel C DNA staining is blue and the bracket marks the approximate boundary of the prd expression stripe. Pav-KLP is detected in stripes of mutant cells expressing Tum (D-F), TumEIE (G-I), TumYRL (J-L), TumPbl (P-R), but not TumPav (M-O). The Pav antibody detects sequestered Pav-KLP protein in the germ line (arrowhead, N) of TumPav-expressing embryos but detects no Pav-KLP in the stripes of TumPav expression. Bar, 5 µm (A-C); 100 µm (D-R).

View larger version (131K): [in a new window]   Fig. 5. Transgene deletions demonstrate that a functional GAP domain is required for Tum cytokinetic function. Tum transgenes with GAP domain deletions UAS-tumEIE (A-E) and UAS-tumYRL (F-L), were expressed in prd stripes in tumDH15 embryos. In all panels, anti-myc antibody shows the transgene localisation in red, tubulin (or F-actin, panel F) localisation is shown in green and DNA in blue. (A-E) When UAS-tumEIE is expressed in tumDH15 embryos, it fails to localise correctly or rescue cytokinesis. Binucleate cells (asterisks, A) are readily detected in stripes of UAS-tumEIE-expressing cells, whereas midbody remnants are rarely found. Cells in the mitotic cycle in these stripes have diffuse TumEIE protein at metaphase (arrow, C), anaphase (arrowhead, C) and telophase (arrow, E). (F-J) When UAS-tumYRL is expressed in tumDH15 embryos, it localises to the cortex equator, but also fails to rescue cytokinesis. Binucleate cells (asterisks, F) are readily detected in stripes of UAS-tumYRL-expressing cells, whereas midbody remnants are rarely found. TumYRL localises to the ends of microtubules at the cell equator at anaphase (arrowhead, F) and this localisation does not change throughout telophase or the subsequent interphase (arrowheads, G,H,I,J). Bar, 5 µm. Grayscale images for all color channels can be viewed in supplementary material Fig. S4.

View larger version (121K): [in a new window]   Fig. 6. Transgene deletions demonstrate that interaction with Pbl and Pav-KLP are required for Tum cytokinetic function. Tum transgenes with a Pav-KLP-interacting domain deletion (UAS-tumPav, A-E) or Pbl-interacting domain deletion (UAS-tumPbl, F-K) were expressed in prd stripes in tumDH15 embryos. In all panels, anti-myc antibody shows the transgene localisation in red, tubulin localisation in green and DNA in blue. (A-E) TumPav protein does not localise with microtubules or rescue cytokinesis. Binucleate cells are readily detected in both expressing and non-expressing stripes of these embryos (A, asterisks) but midbody remnants are not seen. This protein is stable and abundant, but remains diffuse and cortical throughout the mitotic cycle (metaphase, B and C; telophase, D and E). (F-K) Cortex-associated microtubule bundles form in cells expressing TumPbl but cytokinesis fails. TumPbl protein can bind to microtubules and locates at the spindle midzone at anaphase (arrowheads, F,G,H). Rotation of panel H reveals that much of this protein is localised in discrete foci on the cortex (I). Occasional post-metaphase cells viewed along the spindle axis confirm this cortical localisation (arrowheads, J,K). Late-anaphase and telophase cells show partial bundling of microtubules (F) but rarely show evidence of incomplete furrowing. Bar, 5 µm (A-K); 3 µm (I). Grayscale images for all color channels can be viewed in supplementary material Fig. S5.

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