Functional Analysis of Human Kinesin in Cytokinesis Using esiRNA-Mediated RNAi

To investigate the role of the human mitotic kinesin-like protein 1(MKLP1) in mitosis and cytokinesis, E.coli RNase Ⅲ-prepared MKLP1 3′UTR esiRNA was transfected into HeLa cells. quantitive RT-PCR and Western blotting were performed to determine the percentage of the ablation of MKLP1 expression,respectively. Thereafter using FACS analysis, immunofluorescence and time-lapse microscopy to evaluate the cell morphology, mitotic index and percentage of cells at different stages of mitosis and cytokinesis after depletion of the MKLP1 expression, and dynamically observed the phenotypes transformation during mitosis and cytokinesis, to systematically analyse the function of MKLP1. Finally testify the specificity of MKLP1 esiRNA by rescue experiment. These results indicated that MKLP1 esiRNAs specifically and effectively suppress the expression of endogenous MKLP1, which can be compensated by the ectopic expression of MKLP1. MKLP1 proteins were localized the spindle midzone in anaphase and early telophase. During late telophase and the final stage of cytokinesis, MKLP1 was concentrated on the center of the midbody. The depletion of MKLP1 expression result in the severe inhibition of proper formation of the midbody and the completion of cytokinesis. Our results demonstrate that MKLP1 acting as midbody or midzone-associated protein is crucial for the midbody formation and the early telophase to late telophase transition and is essential for cytokinesis.