Functional Study Of Cdc5L In Mitosis

Mitosis is a basic biological process in eukaryotic cells. A series of spatiotemporal control by proteins or protein complex contributes to mitotic fidelity that ensure the genetic material passing to the daughter cells equally. The functional defect of the key factors involved in mitotic regulation may lead to catastrophic events, such as cancer. Therefore, the studies on mitotic process are of great importance, because it will not only uncover the molecular mechanism of mitotic regulation, but also provide more drug targets for the cancer therapies.In order to study the mitotic regulation of M phase, the strategy of this project is to use database searching and siRNA screening for identifying new mitotic regulators. By these means, we finally identified Cdc5L proteins which is involved in M phase regulation. It is well known that Cdc5L (Cell division cycle 5-like protein) function as the regulator of G2 / M phase transition. In addition, Cdc5L is a cell cycle checkpoint protein that physically interacts with ATR and activate the ATR downstream effector, such as Chk1, Rad17. Depletion of Cdc5L by RNA-mediated interference methods results in a defective S-phase cell-cycle checkpoint and cellular sensitivity in response to replication-fork blocking agents. CDC5L also associated with progression and poor prognosis in patients with osteosarcoma. Until now there are no reports about the function of Cdc5L in M phase, but the literature of phosphoproteome showed that Cdc5L could be phosphorylated in M phase, suggesting Cdc5L may be involved in M phase regulation.Our results showed that depletion of Cdc5L in non-synchronized cells leaded to G2 / M phase arrest and cell death. When cell were released from Nocodazol, the depletion of Cdc5L inhibited the mitotic process significantly. At 2h after Noc. release, the cells in control group had a majority of number in G1 phase, but the most of Cdc5L interference cells were in M phase. We further found that Cdc5L played an important role in chromosome congression by Time-lapse assay. The depletion of Cdc5L did not affect spindle assemble, however the kinetochore-microtubule attachment was inhibited. So the spindle was longer, the kinetochore lacked tension, and the mitotic checkpoint was been activated. Since kinetochore protein KNL1 and NDC80 complex is responsible for the kinetochore-microtubule attachment, we then detected the kinetochore localization of KNL1 and NDC80 by immunofluorescence. The results indicated that depletion of Cdc5L weakened the kinetochore localization of KNL1, but did not affect the NDC80 complex. In addition the opposing roles of Aurora kinases and protein phosphatase(PP1) during chromosome congression have long been suggested. Correction of aberrant kinetochore attachment requires a conserved Ser/Thr kinase Aurora B and dephosphorylation of substrates by PP1 is required for stable biorientation of the chromosomes. KNL1 is one of Aurora B kinase substrate, but our WB results showed that Cdc5L did not affect the kinase activity of Aurora B. Due to the cytoplasm expression of Cdc5L without co-localization of kinetochore through the M phase, KNL1 could not be directly recruited by Cdc5L at the kinetochore. Therefore Cdc5L may influence the functions of KNL1 by other proteins or mechanisms.In summary, Cdc5L is a novel M phase regulator that is involved in the kinetochore- microtubule attachment by regulating kinetochore localization and the function of KNL1 and finally ensures M phase process.