Live-cell imaging RNAi screen identifies PP2A-B55a and importin-b1 as key mitotic exit regulators in human cells (Nat Cell Biol, 2010, doi:10.1028/ncb2092)

報告日期: 2010/10/12
報告時間: 15:10/16:00
報告學生: 賴泰佑
講評老師: 徐麗君
附件下載:

Full Text: http://basicmed.med.ncku.edu.tw/admin/up_img/991012-1.pdf

Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells

 

Michael H. A. Schmitz, Michael Held, Veerle Janssens, James R. A. Hutchins, Otto Hudecz, Elitsa Ivanova, Jozef Goris, Laura Trinkle-Mulcahy, Angus I. Lamond, Ina Poser, Anthony A. Hyman, Karl Mechtler, Jan-Michael Peters5 and Daniel W. Gerlich

Nat Cell Biol. 2010 Sep;12(9):886-93. Epub 2010 Aug 15.

 

Speaker: Tai-Yu Lai (賴泰佑)       

Commentator: Li-Jin Hsu, Ph.D.

Date: October 12, 2010 15:10 – 16:00

Place: Room 602, College of Medicine

 

Background

To date, the antimitotic drugs for cancer therapy focus on mitotic checkpoint-induced cell arrest and subsequent apoptosis; however, many cancers can escape cell death by slippage into a tetraploid G1 state. Thus, the inhibition of mitotic exit is the next cancer therapeutic targets.

The mitotic exit depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Previous studies have shown that the protein phosphatases responsible for dephosphorylation of Cdc14 or its substrates in budding yeast, Saccharomyces cervisiae, are Cdc14 phosphatase, but PP1 and PP2A in Xenopus laevis and other species. However, the assays used in these studies were not specific enough for mitotic exit because they may result from defects in early mitosis.

More, since the duration of mitotic exit is too short, the assays of this process in vitro is difficult, which explains why previous RNAi screening of cell division regulators did not annotate mitotic exit phenotypes and the less specificity in previous studies.

 

Objective

Identify the specific human protein phosphatases involving in mitotic exit by the live-cell imaging RNAi screen.

 

Result

  To visually assay mitotic exit in live human cells, the authors established a live-cell imaging system to visualize the metaphase-anaphase transition by a chromatin marker, H2B, and to probe for postmitotic nuclear reassembly by a nuclear import substrate, IBB. Combined the live-cell imaging system and genome-wide RNAi library of human protein phosphatases, the authors identified a trimeric PP2A-B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Further, under the chemically induced mitotic exit assay, the authors found that the PP2A-B55α complex was involving in dephosphorylation of Cdc14 and its substrates. Finally, inportin-β1 has been co-purified with mitotic PP2A-B55α complex and the knockdown of improtin-β1 by RNAi showed the delay of mitotic exit synergistically with PP2A-B55α.

 

Conclusion

PP2A-B55α and importin-β1 are involving in the regulation of mitotic exit in human cells.