JCS02957 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. S2 cell FACs profiles following RNAi of orbit and/or Klp59C, Klp59D and Klp67A. The FACs profiles of S2 cells 72 hours after single RNAi treatment against the kinesin-type depolymerases (A,C,E) or simultaneous with orbit RNAi (B,D,F) are shown. Orbit depletion causes a diminution of 2N cells and an increase in the number of apoptotic cells. Depletion of Klp59C, Klp59D in orbit knockdown cells does not alter the apoptotic cell population. Downregulation of Klp67A alone leads to a marked increase in apoptosis. Double RNAi of Klp67A and orbit exaggerates this effect and the amount of apoptosis increases approximately threefold relative to Orbit depletion alone.

• Movie 1 -

  Movie 1. Spindle formation in a control cell expressing GFP-tubulin. This cell corresponds to that shown in Fig. 3A. The centrosomes are separated from one another during prophase and at prometaphase onset (indicated by the influx of fluorescent tubulin into the nuclear space) a spindle rapidly forms. As the chromosomes move to the spindle equator they form a non-fluorescent ‘shadow’. The spindle reaches a steady state length that it maintains until anaphase onset. Images in this and all subsequent videos are maximum intensity projections of on average seven sections with a 1-μm step size at 20- to 30-second intervals. Playback rate is six frames per second.

• Movie 2 -

  Movie 2. Spindle formation in cells singly depleted of Klp10A or Orbit. This composite video shows cells depleted of Orbit (left hand side) and Klp10A (right hand side) and corresponds to the cells shown in Fig. 3B, C, respectively. In both cells the spindles collapse as the centrosomes come together after prometaphase onset. In the orbit RNAi cell a monopolar spindle forms. Transient multipoles extend outwards from this to form mini-spindles that are unstable and rapidly collapse or change shape leading to a mitotic arrest. After the centrosomes come together in the Klp10A knockdown cell a stable monopolar bipolar spindle forms and the cell ultimately enters into anaphase. See text for details.

• Movie 3 -

  Movie 3. Dual RNAi of orbit and Klp10A prevents spindle collapse and rescues spindle bipolarity. This composite video corresponds to Figs 4A, B and shows two examples of cells that have been treated with RNAi against orbit and Klp10A. Unlike single depletions, the centrosomes in these cells never collapse towards one another and stable, bipolar spindles with a centrosome at each pole are maintained. Note the variation in the time required for progression into anaphase. See text for details.