Supplemental Materials

This article contains the following supporting material:

• Video01 -

  The uncompressed versions of video materials are available at http://bio3d.colorado.edu/mbc/sup_videos/.

  Video 1. The shift to permissive temperature in this and other videos occurred 30 sec after the start of recording (frame 3). After SPBs separation in this nda3 cell, the labeled sister kinetochores are drawn to one of the poles. They then rotate to face the opposite pole and move to the mid-spindle position. Their attachment to opposite sister poles (bi-orientation) is evident from their occasional separation into two distinct spots along the spindle axis.
• Video02 - In this pkl1 Δ nda3 cell the labeled kinetochore bi-orients successfully and expediently.
• Video03 - Similar motions of both kinetochore and poles are seen in the triple deletion cells, pkl1 Δ klp2 Δ dhc1 Δ nda3.
• Video04 - After the labeled sister kinetochores move toward one of the poles in this klp2 Δ nda3 cell, they fail to congress and the spindle does not elongate for the duration of the recording (~ 35 min after kinetochore retrieval). The video sequence pauses briefly to mark the kinetochore arrival at the pole, as determined by analyzing relative pole-kinetochore positions in z-stacks. Note that during all this time the signals from pole-proximal sister kinetochores do not separate significantly from this pole's signal.
• Video05 - In this pkl1 Δ nda3 cell the kinetochore is efficiently retrieved after the temperature shift, but it remains closely associated with the pole during premature anaphase spindle elongation.
• Video06 - EM tomogram of an early pkl1 Δ nda3 spindle (length ~0.75 μm). The MTs nucleated from opposite poles are colored green and red, spindle poles are yellow and the nuclear envelope is gray. Minus ends are shown with orange dots, plus ends with purple dots; ends that lie between the sections are not marked. Occasionally, there were MTs that continued beyond the reconstructed volume; their most distal (plus) ends are marked with green dots.
• Video07 - EM tomogram of pkl1 Δ nda3 spindle. This cell was frozen 4 min later than the one shown in Video 6, and its spindle is longer (1.4 μm). See legend for Video 6 for more details.
• Video08 - Live imaging of dhc1 Δ nda3 cell. After the kinetochore is captured and pulled to the pole, it remains there for an extended time, while fluctuating in position.
• Video09 - Live imaging of dhc1 Δ nda3 cell. The pole-proximal kinetochore is frequently seen nearby the pole during prolonged arrest. When anaphase starts, it briefly lags behind the pole.
• Video10 - EM tomogram of dhc1 Δ nda3 spindle with separated MT bundles. See legend for Video 6 for more details.
• Video11 - Live imaging of pkl1 Δ klp2 Δ dhc1 Δ nda3 cell. The retreival of "lost" kinetochore is somewhat delayed, a characterisitc feature of klp2Δ (Grishchuk and McIntosh, 2006). About 21 min after SPB separation, the kinetochore assumes a pole-proximal position. Anaphase in this triple motor deletion strain starts undelayed, just as frequently seen in the cells lacking the Pkl1p motor.
• Supplemental Figure 1
• Supplemental Figure 2
• Supplemental Figure 3