Oncogene 25:827 (2006)
Toru Miki @ NIH
Authors made a model that Cdk1 phosphorylated Ect2 on T412 generating a priming site for Plk1 PBD binding. Ect2 was shown to be phosphorylated by Cdk1 and Plk1 in vitro. T412 is Cdk1 phosphorylation site. Ect2 has more than one Plk1 phosphorylation site but none has been identified in this paper.
T412A dampened cortical Rho accumulation but T412D caused cortical hyperactivity during cell division. It suggests T412 plays an important role for regulating Rho activity. Plk1 interacted with wildtype Ect2, T412D, but not T412A. Plk1 binding Ect2 is important for this paper's novelty. However, authors did not show any direct evidence that Plk1 interacts with phosphor-T412. A direct phosphopeptide binding experiment will be perfect for this issue. In David Pellman's yeast Cdc5 and Rho GEFs paper, David mentioned Plk1 interaction cannot be rescued by phosphor-mimic mutant. So Plk1 should not interact with T412D if T412 serves as a priming site for PBD.
Although Ect2 contains a PH domain, it appears that the main functional localization is on midzone/midbody. It is different to the Science paper from David Pellman's lab. However, it is still very interesting to figure out whether Plk1 phosphorylates Ect2 in vivo. Cdk1/cyclin B degradation is an essential step for cytokinesis entry. Therefore the T412 phosphorylation must happens before cytokinesis if it is essential for fully activating Rho.
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4 comments:
真認真, 跟你拼了XD
我也要開始認真唸書....
只是要分心唸亂修的發育生物學
還真是有點傷腦筋咧
問你一個問題
你最長可以 follow活細胞多久阿
我了不起才20-24小時
這樣很難做比較精巧的實驗
-___-
With CO2 chamber at 37'C, we can image as long as we want. Without CO2 chamber, there will be several obstacles for long-time imaging.
PH in medium changes very fast without CO2 supply. It could be overcame by using CO2-independent medium. However, I found CO2-independent medium somehow causes a mild microtubule disorganization in monopolar mitotic cells. Other people in my lab never complain about it. So that is probably my cell's problem, or just because monopolar cell spindles are more sensitive than bipolar spindles.
Evaporation is also a problem. In our microscope room, the evaporation is really strong. Put some mineral oil on medium can greatly improve this problem.
Temperature is also an issue. Outside the chamber, cells usually are more sick at 37'C than lower temperatures. We usually use 35'C instead and cells are happier.
For my own experiment, I need to slow a fast reaction down and manipulate multiple small molecules during the imaging. I just leave the cell at room temp without mineral oil. Cells are good only within 2 hrs. But with chamber, the longest period of time so far is 4 days.
Well, It seems money talks. We will have a CO2 chamber in the "future".
Before that, we tried several tricks to overcome the evaporation and pH issue. Actually I have succeeded in long-time imaging couple times for more than 24 hrs. But, recently, I tried to shift my experiment from HeLa to hRPE; the cells die almost immediately. That's why I ask.
I used HEPES to buffer medium and sealed the chamber with inert grease to avoid evaporation. We right now suspect the grease for killing my hRPE cells.
But the suggestion of temperature may be worthy to test. Thanks.
Acturally we don't have a CO2 chamber either. We need to go to the departmental imaging facility for it. In case you don't remember, it is supervised by a very-difficult-to-deal-with person named Jennifer Waters. :)
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