Hi All,
Has anybody with experience in inducing mitosis by any chemical reagents..? My main aim is to activate APC/cdc20 complex along with the start of M phase.
Currently only method I am following is to arrest the cells (by RO-3306) at G2-M boarder in order to synchronization at the beginning of M-phase and then release the cell from the inhibitor. Then the cells start entering to mitosis. Can this method suffice to induce APC activation as well..?
If anybody can suggest any other protocol.
You are most welcome..!!
Thanks
Dear Sahu,
Ubiquitin-mediated protein destruction plays a critical role in regulation of cell cycle progression, and is a particularly effective method for promoting unidirectional progression in the cell cycle because of its irreversibility.
Dear Walid,
Thanks for your suggestion.
But did not really understand your point.
Can you please elaborate it..?
Thanks.
Dear Indrajit,
It would probably be helpful if you share with us what you intend to do: cell type, bulk biochemistry (requires many cells and decent synchrony), immunofluorescence (fewer cells that can even be selected by additional markers) etc.
Your current approach will typically result in ~60% of cells entering mitosis within 20 min of release from RO3306. As cells enter mitosis, Cdk(1) activity remains high until all chromosomes are bioriented in the metaphase plate and correctly attached to microtubules. During this time, any ubiquitin ligase activity of the APC-cdc20 is inhibited by the mitotic checkpoint complex (Mad2/BubR1/Bub3+/-cdc20) on the one hand, and APC-cdh1 inhibition by Cdk1 activity. The mitotic checkpoint complex requires Mps1 kinase for its formation.
Normally, the mitotic checkpoint complex stops being formed (or is perhaps actively disassembled) once all chromosomes are properly attached. This is the metaphase-anaphase transition at which the APC becomes active and starts destroying cyclin B (and many other proteins). If you give your cells ~35 minutes after RO3306 washout, a varying number of cells will be in this state.
If you were interested in getting a bunch of cells that are biochemically in early anaphase, you can first arrest them in nocodazole for 4-16 hrs, which destabilizes microtubules and arrests cells in prometaphase. You could separate these from non-mitotic cells by a mitotic shake-off (bang the dish to dislodge the mitotic cells and collect these by washing). These will still be in a state of high Cdk1 activity and inhibited APC-cdc20 (and no real spindle). If you now treat these cells with an Mps1 inhibitor such as reversine, the mitotic checkpoint complex stops being formed despite all chromosomes being unattached and the APC-cdc20 complex becomes active.
Obviously, this is rather lousy if you want to look at anaphase spindles.
Alternatively, you might arrest cells in nocodazole first, wash out nocodazole while washing in MG132, allow cells to form spindles, then wash out MG132.
Hope this helps!
Dear Indra A Shaltiel,
Thank you so much for such a detail explanation..!!
My main aim is to express various truncated form of Cyclin-B (which do not hinder mitosis progression) and get ubiquitinated by APC/cdc20 complex. Then to follow their degradation by 26S proteasomes.
As you correctly said about ~30 min post RO3306 washout the APC complex will be activated. So should I follow the degradation for up to 2hr then or more..?
(Because I did not see any significant degradation of the endogenous CyclinB by 1hr)
What your experience says..??
Thanks in advance.
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