A countdown clock in mitotic prophase: design logic for dual pathway mitosis

Background / Purpose:

The purpose of the prophase pathway of mitosis, in which the two centrosomes migrate to opposite sides of the nucleus ahead of nuclear envelope breakdown, is to increase the fidelity of chromosome segregation. We find that the ~50% of HeLa cells that successfully separate their centrosomes during prophase do indeed exhibit ~10-fold fewer chromosome segregation errors, and that centrosome migration is a race against the clock, with the limited success rate being the result of the limited (9.2 min) time available for centrosome migration, between the start of prophase (loading the kinesin-5 motor to the centrosomes) and nuclear envelope breakdown. Prophase centrosome separation reduces segregation errors, but is, as we show, slow and stochastic, so that limiting the time available for centrosome racing may represent for the cell an optimized strategy that trades off the need for accurate chromosome segregation against the need for rapid mitosis.
It is known that kinesin-5 motors drive the antiparallel sliding of centrosome-attached microtubules that causes the centrosomes to move to opposite sides of the nucleus in mitotic prophase. Using GFP-kinesin-5 and mCherry tubulin, we show that the “green light” afforded by kinesin-5 loading to the centrosomes represents both a readout of the status of the mitotic clock and a fiducial marker for the onset of prophase. Use of this fiducial green light revealed that the mitotic clock sanctions a time window of 9.2 minutes between prophase onset and nuclear envelope breakdown during which the centrosomes can attempt to separate.
We used:

• HeLa cells, stably transfected with mCherry tubulin and treated with RNAi eGFP-full length kinesin-5.


• EI III, a monastrol-like inhibitor, to modulate the activity of kinesin-5.


• Various mitotic kinase inhibitors to show that kinesin-5 loading is controlled by the mitotic clock.


• Labeled chromatin.


• Time lapse live cell epifluorescence microscopy using the Deltavision.

Main conclusion:

• The loading of kinesin-5 to the centrosomes marks the opening of a time window, during which the centrosomes race to separate by kinesin-5 driven microtubule sliding before prophase is brought to an end by nuclear envelope breakdown. About half of the HeLa cells succeed in fully separating their centrosomes ahead of nuclear envelope breakdown.


• Success at separating the centrosomes in prophase reduces by ~10-fold the probability of subsequent chromosome segregation errors.


• Using the mitotic clock to specify a limited (9.2 min) time window between kinesin-5 loading (start of prophase) and nuclear envelope breakdown (end of prophase) may represents an optimised strategy by which cells trade off the need for rapid spindle assembly against the need for accurate chromosome segregation.

Our work reveals prophase centrosome separation as a race against the (mitotic) clock. In HeLa cells, success in this race improves the fidelity of chromosome segregation by ~10-fold. Now that we know why prophase centrosome separation is a good thing, it will be important to ask, what is it about the mechanism of centrosome separation that makes it apparently so intrinsically slow and difficult that only 50% of cells manage to completely separate their centrosomes ahead of nuclear envelope breakdown?

Disclosures:

Funded by Marie Curie Cancer Care.