Functional Delineation Of Azu-1 And PinX1 In Mitosis

In animal cells, centrosome, also called microtubule organization center (MTOC), plays an important part in plasticity of cell polarity, assists with introcellular transporting and participates in the management of mitotic behaviors. Accurate replication and segregation of centrosomes are the premises of daughter cell division, avoiding false separation of chromosomes. Thus, centrosome abnormalities can lead to chromosome instability and cancer.As a member of Aurora serine/threonine kinase family, Aurora A plays a significant role in maturation of centrosomes and assembly of bipolar spindles related to its centrosome localization during mitosis. Suppression of Aurora A causes errors when centriole pairs separate, or chromosomes congress to the spindle equator. Recently, Xenopus and Drosophila transforming acidic coiled coil (TACC) proteins have been found to be the substrates of Aurora A. After phosphorylation by Aurora A, TACC proteins are recruited to centrosomes, and then contribute to modulating centrosomal microtubule stability with microtubule associated proteins. Three TACC proteins (TACC1, TACC2, TACC3) exist in human, and TACC3 has been proved to be phosphorylated by Aurora A. However little is known about TACC2. In this study, we focus on anti-zuai-1 (Azu-1), which is identified as an isoform of TACC2. We have demonstrated that Azu-1 interacts with Aurora A through its N-terminal region and co-localizes with Aurora A at centrosomes in mitotic HeLa cells. Moreover, Azu-1 can be phosphorylated by Aurora A kinase and the phosphorylation is essential for centrosomal localization of Azu-1.On the other hand, most of the important things, comprising DNA's replication, interaction of chromosomes with microtubules and accurate segregation to the daughter cells, are monitored by the cell cycle checkpoint in the procession of cell division. When error occurs, spindle assembly checkpoint will be activated, which assures that kinetochore could align precisely on the equatorial plate and cell cycle continues from metaphase to anaphase. Spindle assembly checkpoint warrants the exact separation of chromosomes. Mechanisms concerning these cell cycle events are conserved from the simple mono-cell eukaryote to the complicated metazoan.PinX1 is a potent telomerase inhibitor in interphase; however, its function in mitosis is not well documented. Here we show that PinX1 is essential for faithful chromosome segregation. Deconvolution microscopic analyses show that PinX1 localizes to nucleoli and telomeres in interphase and relocates to the periphery of chromosomes and outer plate of the kinetochores in mitosis. Our deletion analyses mapped PinX1's kinetochore localization domain to the central region and its chromosome periphery localization domain to the C terminus. Interestingly, the kinetochore localization of PinX1 is dependent on Hec1 and CENP-E. Our biochemical characterization revealed that PinX1 is a novel microtubule-binding protein. Our real-time imaging analyses show that suppression of PinX1 by small interference RNA abrogates faithful chromosome segregation and results in anaphase chromatid bridges in mitosis and micronuclei in interphase, suggesting an essential role of PinX1 in chromosome stability. Furthermore we report that PinX1 interacts with Nucleolin, a chromosome periphery protein, through its C-termini. Deconvolution microscopic analyses show PinX1 mainly co-localizes with Nucleolin at chromosome periphery in prometaphase. Moreover, depletion of Nucleolin abolishes chromosome periphery localizations of PinX1, suggesting a functional interrelationship between PinX1 and Nucleolin. Importantly, repression of PinX1 and Nucleolin abrogates chromosome segregation in real-time mitosis, validating the functional importance of PinX1-Nucleolin interaction. We propose PinX1 is recruited to chromosome periphery by Nucleolin and a complex of PinX1 and Nucleolin is essential for faithful chromosome congression.