Video data from "A Cell Cycle Timer for Asymmetric Spindle Positioning"

Erin K. McCarthy Campbell, Adam D. Werts, Bob Goldstein

PLoS Biol 7(4): e1000088 (pdf)



These experiments demonstrated that APC-dependent negative regulation of CDK, long known to function as a mitotic progression timer, serves a second role as a timer for spindle displacement during an asymmetric cell division. This timing mechanism ensures that spindle assembly is completed before spindle components begin to move away from the center of the cell.

asymmetric spindle checkpoint





Video S1. The Mitotic Spindle Begins to Shift Approximately 10 s after the Completion of Chromosome Congression.
Movie of the embryo from Figure 1B–D expressing histone H2B:GFP and gamma-tubulin:GFP, and a dynamically annotated version of the corresponding graph. Dotted line in the movie marks 50% embryo length. As the fluorescence ratio in the center of the spindle to just outside this region peaks at metaphase, displacement of the spindle with a compact metaphase plate is evident in the embryo and the graph. Light blue datapoints in this graph indicate the center of the two chromatin masses after anaphase.





Video S2.
Disruption of Proteasome Activity Delayed Both Spindle Displacement and Anaphase Onset. Three cases of each treatment: wild-type untreated (top three), rpt-6(RNAi) (middle three), and c-LβL (bottom three). Small regions of each embryo are shown, each through most of the mitotic spindle. The red line is 50% embryo length, and each movie starts at nuclear envelope breakdown.




Video S3. Premature CDK Inactivation Caused Premature Spindle Displacement and Anaphase Onset. Three cases of each, untreated (top three) and flavopiridol-treated (bottom three). Small regions of each embryo are shown, each through most of the mitotic spindle. The red line is 50% embryo length, and each movie starts at nuclear envelope breakdown. Blank frames are shown at time of flavopiridol-treatment.




Video S4. Anaphase Timing Is Unaffected When Spindle Displacement Is Prevented. WT is wild-type control. The vertical line is 50% embryo length, and each movie starts at nuclear envelope breakdown. Spindle displacement was prevented by par-2 RNAi or gpr-1/2 RNAi.




Video S5. Flavopiridol Treatment as Nuclear Envelope Breakdown Began Resulted in Chromosomes Being Spread over a Significantly Wider Area Than Normal at the Time of Spindle Displacement. An embryo laser-permeabilized to flavopiridol as nuclear envelope breakdown began (right). Spindle displacement toward the posterior (top) and anaphase both occur before chromosome congression is complete in the flavopiridol-treated embryo. The embryo in the right is the bottom embryo among the flavopiridol-treated embryos in Figure 6A. The embryo on the left was laser-permeabilized to flavopiridol later than the embryo on the right was, and chromosome dynamics appear less affected as expected.




Video S6. Flavopiridol Treatment Directly after the Start of Nuclear Envelope Breakdown Resulted Sometimes in Individual Chromosomes Failing to Associate with a Metaphase Plate by the Time of Premature Spindle Displacement. As in the previous video, but this is a treated embryo alone, with an apparently less severe defect. One chromosome is not associated with the metaphase plate during spindle displacement and anaphase. This embryo is also shown as the center embryo among the flavopiridol-treated embryos in Figure 6A.



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