Study On The Purification Of Mitotic Spindle And Midbody NLP And KIF4A May Be Involved In Regulating Midbody Formation.

The mitotic spindle and midbody are both microtubule-based temporary structure during cell growth and play an essential role in mitosis. Mitosis is the most basic life activities of eukaryotic cell, and failures in mitosis can lead to birth defects, leukemias, and tissue-specific tumors.Many factors that regulate mitotic spindle function and cell division have been identified using genetic and biochemical methods over the past forty years. Recently, genomic and proteomic screens have increased the growing number of mitotic and cell division factors, yet many of the components necessary for mitotic spindle function and cell division still remain unknown. In order to understand how this macromolecular machine drives mitosis, the field has started to catalog all components in mitotic structures and construct networks of protein interactions. Additionally, the comparative analysis of spindle proteome with the midbody proteome will lay a foundation for revealing the cytokinesis mechanism. And the proteins in the midbody are considered as the key to understand cytokinesis.In the first part of the dissertation, we enriched mitotic spindle or midbody of cells through the cell cycle synchronization method. Then we use low permeability swelling and glycerol gradient centrifugation to extract spindles and midbodies. By Western Blot and immunofluorescence staining, the extracts were further identified as mitotic spindles and midbodies. In this study, we successfully purified mitotic spindles and midbodies from synchronized HeLa cells. This will be of great significance to the study of molecular regulatory mechanisms of mitosis and tumorigenesis.The second part of the dissertation was the preliminary study of NLP and KIF4A regulating midbody formation. NLP protein is a very important protein for mitosis. Previous studies have reported that NLP is required for centrosomes maturation and spindle formation in mitosis, also contributes to the accomplishment of cytokinesis. KIF4A protein is a very important kinesin for mitosis, and involved in chromosomes alignment, transport PRC1protein to spindle midzone. Western Blot results showed that the expression of NLP protein was positively correlated with that of KIF4A protein. With immunofluorescence staining, the two proteins were identified to localize to the midbody. Additionally, the defection of NLP or KIF4A by siRNA could induce morphological changes of midbodies and formation of binucleated/multinuclear cells. So our studies give some clues that NLP and KIF4A in the midbody probably have collaborative functions to the regulation of midbody formation.