Regulation Of Microtubule Organization And Microtubule-Related Cellular Activities By The Centrosomal Protein Cep70

The centrosome, composed of a pair of barrel-shaped centrioles surrounded byelectron-dense pericentriolar material, is the major microtubule-organizing center inanimal cells. In addition to its role in the nucleation and organization of microtubules,the centrosome is also critical for the orchestration of cell cycle progression. Toensure bipolar organization and geometric precision of the mitotic spindle, thecentrosome serves as a cellular hub where several key regulators convene to mediatevital interactions that control spindle assembly. The centrosome may also serve toregulate other fundamental cellular functions, such as cell polarity and motility.Cep70, a candidate centrosomal protein of70kDa, was originally discovered ina proteomic study of the human centrosome. Recently, the biological function ofCep70in different species is beginning to be unraveled. The zebrafish homolog ofCep70has been shown to contribute to the assembly of cilia by determination of thelength of the axoneme. In addition, the Chlamydomonas procentriole protein CRC70,a member of the conserved Cep70family, acts as a scaffold for the assembly of thecentriole precursor. However, the localization and functions of Cep70in mammaliancells remain unknown.In this study, we report the molecular characterization and functional analysis ofa novel centrosomal protein, Cep70, in mammalian cells. Our data show that Cep70localizes to the centrosome throughout the cell cycle and binds to the key centrosomalcomponent,-tubulin, through the peptide fragments that contain the coiled-coildomains. Our data further reveal that the centrosomal localization pattern of Cep70isdependent on its interaction with-tubulin. Strikingly, Cep70plays a significant rolein the organization of both preexisting and nascent microtubules in interphase cells.Besides, Cep70promotes microtubule assembly in vitro by increasing microtubuleelongation. In addition, Cep70is necessary for the organization and orientation of thebipolar spindle during mitosis. These results thus report for the first time theidentification of Cep70as an important centrosomal protein that interacts with -tubulin and underscore its critical role in the regulation of mitotic spindle assembly.Centrosomal proteins are important for the organization of microtubules inanimal cells and regulate diverse microtubule-mediated cellular activities such as cellmigration, a critical step in angiogenesis. However, the participation of these proteinsin angiogenesis, which involves vascular endothelial cell migration from preexistingblood vessels, remains elusive. Here, we demonstrate that Cep70is necessary forangiogenic response in mice. This protein is also required for tube formation andcapillary sprouting in vitro from vascular endothelial cells. Wound healing andtranswell assays reveal that Cep70plays a significant role in endothelial cellmigration. Depletion of Cep70results in severe defects in membrane ruffle formationand centrosome reorientation, indicating a requirement for this protein in cellpolarization. In addition, Cep70is critically involved in microtubule rearrangement inresponse to the migratory stimulus. Our data further demonstrate that Cep70isimportant for Cdc42and Rac1activation to promote angiogenesis. These findingsthus establish Cep70as a critical regulator of the angiogenic process and emphasizethe significance of microtubules for vascular endothelial cell migration andangiogenesis.In conclusion, we describe the characterization of Cep70as a centrosomalprotein and demonstrate its localization to the centrosome throughout the cell cyclevia interaction with-tubulin. We also present evidence that Cep70is a criticalregulator for the organization of radial microtubule arrays and the spindle apparatus.In addition, Cep70is implicated in angiogenesis by regulating vascular endothelialpolarization and migration.