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Fig. 2. Cdc8 localises to actin filaments throughout the cell division cycle. (A) Cdc8 immunofluorescence staining of the wild-type strain DPM219 using Cdc8 antiserum. Panels are numbered (i-vii) to show successive cell cycle stages. During G2 (i, ii and iii) Cdc8 localised to filaments that extend to the full length of the cell. Upon entry into mitosis (iv) Cdc8 filaments extend around the cell equator as Cdc8 filaments are incorporated into the CAR, with signal intensity increasing throughout mitosis (v and vi). During contraction, Cdc8 filaments extend out of the CAR into the cytoplasm (vii) to be incorporated into filament structures in the two daughter cells. (B) Co-staining of Cdc8 (green), microtubules (red) and nuclear material (blue) with Cdc8 antisera, TAT1 antibody and DAPI, respectively, revealed that Cdc8 filaments incorporated into a medial ring structure prior to metaphase. (C) A z-series through a wild-type cell subjected to Cdc8 immunofluorescence staining. 0.25-µm serial sections extend from the bottom (0 µm) to the top (2.0 µm) of the cell. Filaments can be seen to extend to the full length of the cell. (D) Simultaneous staining with Cdc8 serum (left panel, green in right panel) and Rhodamine-phalloidin (middle panel, red in right panel) demonstrate that Cdc8 colocalises with actin. (E,F) Left panels show Cdc8 localisation and right panels show DAPI phase images of the same cells. Cdc8 filaments were absent in cdc3-124 cells incubated at the restrictive temperature (E), and in wild-type cells treated with 10 µM Latrunculin A (F), demonstrating Cdc8 filament integrity in vivo requires F-actin. (G,H) Cdc8 localised (green) to the CAR and interphase filaments (arrows) in cells lacking microtubules. DAPI staining (blue) revealing nuclear positioning and fragmentation defects synonymous with microtubule depolymerisation brought about by either nda2-km52 cells (G), or treating wild type cells with carbendazim (H). Bars, 2 µm.
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54kDa, the intensity of which was comparable to the endogenous Cdc8 band. The same GFP-Cdc8 band was seen when the membrane was probed with anti-GFP antibody (B). (C) The same membrane probed with TAT1 to demonstrate equal loading of protein. A population of DPM126 cells were synchronised using transient block and release of the cdc25-22 allele. Two cell cycles were followed and samples were taken every 20 minutes for western blot analysis and for scoring the appearance of actin rings and septation index at each time point. Positions of molecular mass markers (in kDa) are indicated. (D) The proportion of cells possessing cytokinetic actomyosin rings (O) or a septum (+) at each time point over the two cell cycles. A western blot of whole cell extracts from each time point was probed with both Cdc8 anti-sera (E) and TAT1 antibody (F). Cdc8 migrates as a constant doublet throughout the cell cycle. Quantification of the ratio of TAT1 to Cdc8 doublet signals were analysed from three independent experiments and revealed no fluctuation in Cdc8 levels as cells progress through the cell cycle.
) dimers, as measured by quantitative analysis of supernatant and pellet co-sedimentation gels (A), and calculated using the Hill equation (see Table 1). (C) Supernatant and pellet co-sedimentation gels of unacetylated and endogenous Cdc8. The faster migrating endogenous Cdc8 band associated with actin in the pellet, while the slower migrating band was the prominent form in the supernatant. (D) Endogenous Cdc8 purified from actin co-sedimentation pellets from C migrated more slowly than unacetylated Cdc8 purified from E. coli.
