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Files in this Data Supplement:
Fig. S1. Anaphase B spindle length determination. An example of a klp67A mutant cell during anaphase B. The maximum intensity projection of the six b-tubulin-EGFP sections are shown in red and superimposed on the centre-most DIC section (blue). A seven segment white line has been drawn between the centrosomes and through the spindle’s long axis along the midline between the sides of the spindle envelope (outlined in black and labelled SE). By determining the length of this line it is possible to monitor spindle elongation in the absence of centrosome movement.
Fig. S2. Distribution of Klp67A in primary spermatocytes from various genetic backgrounds. (A-C) Localisation of Klp67A in wild-type Oregon R cells. Microtubules are shown in green, Klp67A in red and DNA in blue. (A) During metaphase Klp67A is excluded from the regions of the cell occupied by spindle microtubules. It is also seen in punctae that correspond to kinetochores (arrowheads). (B-C) Following anaphase onset Klp67A redistributes to the midzone of the central spindle and remains associated with the midbody after cytokinesis. (D) Polo kinase is not required for Klp67A localisation at the post anaphase spindle equator. polo1/polo10 mutant testes were fixed and stained for the distribution of Klp67A (red), microtubules (green) and DNA (blue). Although no central spindle forms in the polo mutant, Klp67A still concentrates at cell’s equator. (E) Pavarotti kinesin-like protein and Klp67A colocalise on central spindles. Spermatocytes expressing GFP-tagged pavarotti (green) were fixed and stained with antibodies to Klp67A (red) and DNA (blue). Both kinesin-like proteins colocalise on the central spindle. However, the Klp67A signal is slightly more diffuse. Note how pavarotti localises to the ring canals but Klp67A does not. Bars, 10 mm.
Movie 1. Anaphase B in a wild-type primary spermatocyte expressing b-tubulin-EGFP. The single centre-most DIC section is shown on the left and on the right is the maximum intensity projection of the six fluorescent optical sections. Time is in minutes relative to anaphase onset. Playback rate is five frames per second. This format is used in all subsequent supplementary movies. This is the same cell as shown in Fig. 2A. See text for details.
Movie 2. Anaphase B in a klp67A mutant primary spermatocyte expressing b-tubulin-EGFP. The centrosomes in this cell do not move as the spindle elongates resulting in its buckling outwards. The cell corresponds to that depicted in Fig. 2B. See text for more information.
Movie 3. Central spindle formation in a wild-type primary spermatocyte. See Fig. 4A and text for details.
Movie 4. klp67A mutants fail to form stable central spindles. During metaphase the spindle and asters are abnormally robust. However, on entry into anaphase the microtubules become less stable and those in the cytoplasm as well as within the spindle envelope rapidly deteriorate. See Fig. 4B and text for details.
Movie 5. Ectopic furrows form cytoplasts in klp67A mutants. This cell corresponds to that shown in Fig. 5. See text for more information.
Movie 6. Interior central spindles can induce cleavage furrows. This cell is also depicted in Fig. 6. Note how the ectopic furrow only initiates after interior central spindle microtubules come in proximity to the cortex. See text for details.
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