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 tum^DH15 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.

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 tum^DH15 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 tum^DH15 (D) embryos. Diaphanous is cortical throughout the cell cycle, but concentrates at the midzone cortex of wild-type telophase cells (arrowheads, C). In tum^DH15 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 tum^DH15 (G) embryos. At anaphase, Pbl-GFP is concentrated in the midzone cortex in normal cells (arrowheads, E,F) but remains cortical throughout mitosis in tum^DH15 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 tum^DH15 (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 tum^DH15 (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.

Elevated Pav-KLP protein levels occur in stripes of cells expressing all but the UAS-tum^ΔPav 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), Tum^ΔEIE (G-I), Tum^ΔYRL (J-L), Tum^ΔPbl (P-R), but not Tum^ΔPav (M-O). The Pav antibody detects sequestered Pav-KLP protein in the germ line (arrowhead, N) of Tum^ΔPav-expressing embryos but detects no Pav-KLP in the stripes of Tum^ΔPav expression. Bar, 5 μm (A-C); 100 μm (D-R).

Transgene deletions demonstrate that a functional GAP domain is required for Tum cytokinetic function. Tum transgenes with GAP domain deletions UAS-tum^ΔEIE (A-E) and UAS-tum^ΔYRL (F-L), were expressed in prd stripes in tum^DH15 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-tum^ΔEIE is expressed in tum^DH15 embryos, it fails to localise correctly or rescue cytokinesis. Binucleate cells (asterisks, A) are readily detected in stripes of UAS-tum^ΔEIE-expressing cells, whereas midbody remnants are rarely found. Cells in the mitotic cycle in these stripes have diffuse Tum^ΔEIE protein at metaphase (arrow, C), anaphase (arrowhead, C) and telophase (arrow, E). (F-J) When UAS-tum^ΔYRL is expressed in tum^DH15 embryos, it localises to the cortex equator, but also fails to rescue cytokinesis. Binucleate cells (asterisks, F) are readily detected in stripes of UAS-tum^ΔYRL-expressing cells, whereas midbody remnants are rarely found. Tum^ΔYRL 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.