Study On The Role And Mechanism Of Abi-1 During Cytokinesis

Objective :Cytokinesis is a critical process at the end of cell division. Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independent daughter cells. This indicates that cytokinesis is a checkpoint for the right cell division. 1A). The contractile ring is a network of actin and myosin filaments。The formation of contractile ring is essential for the onset and achievement of cytokinesis. However, the molecules and signal pathways responsible for these processes have remained elusive. Recent studies have provided insights into that Abi-1 regulates the reorganization of actin cytoskeletal and is involved in cell moving. Nevertheless, it is unknown whether Abi-1 participates in the formation of contractile ring. Here, we try to analyse the key role of Abi-1 during cytokenesis and on cell growth.Methods:1. Using confocal microscopy, we examined the subcellular localization of endogenous Abi-1 and actin in NIH3T3 cells at different stages of late mitosis by immunofluorescence staining,from anaphase to cytokinesis.2. Abi-1-siRNA plasmid stable transfected NIH3T3 cell line was constructed. Abi-1-depleted cells were immunofluorescence stained and the process of cytokinesis was observed by confocal microscopy.3. We tested the cell cycle and apoptosis by PI and API using FCM, the cell proliferation by Ki67 stain, the clone formation in soft agar and tumor growth experiment in Abi-1-depleted cells comparing the parent NIH3T3 cells.4. The levels of CDK1 and Tyr15 protein were detected by Western Blotting. Results:1. Our study shows that endogenous Abi-1 colocalized with cortical actin in the cellular membrane and appeared to concentrate in the cleavage furrow to the end of ring contraction. At the end of telophase, when the two daughter cells began to separate, Abi-1 was also detected in the midbody. In the G1 phase of the cell cycle, Abi-1 was diffusely scattered throughout the cytoplasm and did not exhibit predominant colocalization with actin.2. The depletion of Abi-1 resulted in aberrant localization of actin and failed cleavage furrow and contractile ring formation in anaphase of cell division. Ctokinesis was blocked. RNA interference–mediated silencing of Abi-1 in cultured cells results in the formation of giant and multinucleate cells. Quantitative analysis revealed that Abi-1 depletion significantly increased the formation of multinucleated cells (27.68%), compared with the parental NIH3T3 cells (2.93%).3. The cell growth was inhibited in Abi-1-depleted cells, including G1 arrest, decreased Ki67 expression, lower formation of clone and failure growth in athymic mouse.4. The decreased levels of CDK1 Tyr15 proteins tested by western blotting revealed that the activity of CDK1 was higher in Abi-1 depleted cells.Conclusions:1. Abi-1 may regulate the reorganization of actin cytoskeleton and involve in the formation of contractile ring. This suggest that Abi-1 serve as a master controller of cytokinesis.2. Abi-1-depletion leads the failure of cytokinesis and multinucleation.3. The level of Abi-1 affects cell growth. Abi-1-depletion negatively regulates cell growth.4. The regulation on cytokinesis and cell growth of Abi-1 may completed through CDK1.