Regulation Of Microtubule Assembly And Microtubule-related Cellular Activities By The Tumor Suppressor CYLD

The microtubule cytoskeleton plays an important role in many cellular activities, such as cell shaping, intracellular trafficking, cell division, and cell motility. Microtubules are intrinsically dynamic polymers and the dynamic property of microtubules is critical for many of their cellular functions. The structure and function of microtubules are regulated exquisitely in cells by a repertoire of microtubule-binding proteins, including proteins that regulate the assembly and organization of microtubules and motor proteins that mediate the transport of organelles and vesicles. Alteration of microtubule assembly and/or stability by abnormal expression of microtubule-binding proteins or by drug treatment can result in serious phenotypes such as defects in cell motility. cell cycle arrest, or even cell death.The tumor suppressor for human familial cylindromatosis (CYLD) is a protein that consists of 956 amino acid residues with three CAP-Gly domains in the amino terminus. In addition, a deubiquitinase domain exists in the carboxyl terminus of CYLD conferring its activity to deconjugate lysine-63-linked polyubiquitin chains from target proteins. CAP-Gly domains exist in a number of microtubule-binding proteins and are responsible for their association with microtubules. However, it remains elusive whether CYLD interacts with microtubules and, if so, whether the interaction is mediated by the CAP-Gly domains. In this study, immunofluorescence microscopy shows that endogenous CYLD colocalizes with the microtubule cytoskeleton in cells. Microtubule cosedimentation assays further demonstrate a direct interaction of CYLD with microtubules in vitro. In addition, microtubule cosedimentation assays reveal that the first CAP-Gly domain of CYLD is mainly responsible for its interaction with microtubules. By GST pulldown assays, we find that CYLD interacts with the microtubule subunit tubulin. Knockdown of cellular CYLD expression dramatically delays microtubule regrowth after depolymerization, indicating an activity for CYLD in promoting microtubule assembly. By tubulin turbidity assay, we find that CYLD promotes tubulin polymerization into microtubules and stabilizes microtubules against dilution-induced depolymerization. Furthermore, tubulin turbidity and microtubule sedimentation assays both reveal that CYLD enhances tubulin polymerization into microtubules by lowering the critical concentration for microtubule assembly. In addition, we identify by wound healing assay a critical role for CYLD in mediating cell migration and find that its first CAP-Gly domain is required for this activity. Thus CYLD joins a growing list of CAP-Gly domain-containing proteins that regulate microtubule dynamics and functions.Although CYLD was initially identified as a tumor suppressor, it has recently been implicated in diverse physiologic processes, such as immune response. inflammation, osteoclastogenesis. and cell cycle progression. In this study, we have investigated the involvement of CYLD in angiogenesis, a process tightly regulated by pro- and anti-angiogenic factors and requires the migration of vascular endothelial cells from preexisting blood vessels. We find that knockdown of CYLD expression significantly impairs angiogenesis in vitro in both matrigel-based tube formation assay and collagen-based three-dimensional capillary sprouting assay. The effect of CYLD on angiogenesis is also studied in vivo. We find that addition of CYLD siRNA or anti-CYLD antibody significantly blocks vascular growth into the angioreactors implanted in mice. We have also investigated the molecular mechanisms that underlie the function of CYLD in angiogenesis. Cell spreading and wound healing assays reveal that CYLD regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. Examination of membrane ruffling at the leading edge of migrating cells shows diminished membrane ruffling in CYLD-knockdown cells, providing further evidence for the involvement of CYLD in vascular endothelial cell migration. In addition, we find that silencing of CYLD dramatically decreases microtubule dynamics in endothelial cells and inhibits endothelial cell migration by blocking the polarization process. To gain more mechanistic insight into how CYLD mediates endothelial cell migration and angiogenesis, we have investigated the involvement of the Rho family GTPases in these processes. Our data identify Rac1 activation as an important factor contributing to the action of CYLD in regulating endothelial cell migration and angiogenesis. These results thus uncover a previously unrecognized role for CYLD in the angiogenic process and provide a novel mechanism for Racl activation during endothelial cell migration and angiogenesis.CYLD has been shown to regulate cell cycle progression, but the precise molecular mechanisms remain to be elucidated. Microtubules undergo dramatic rearrangement at the onset of mitosis, and exquisite microtubule dynamics are required for mitotic spindle assembly and chromosome segregation. The finding that CYLD regulates microtubule dynamics suggests that CYLD may play an important role in mitosis. To investigate this hypothesis, we have studied the effect of CYLD on spindle assembly. We find that CYLD knockdown results in spindle misorientaion, indicating a critical role for CYLD in spindle positioning. Immunofluorescence microscopy reveals that CYLD mediates the interaction of astral microtubules with the cell cortex. By GST pulldown assay. we find that CYLD interacts with the microtubule tip-associated protein EB1. a known linker between microtubule plus ends and the cell cortex. Immunofluorescence microscopy further shows that CYLD and EB1 colocalize at the ends of some astral microtubules. In addition. CYLD can form dimers in cells, and the dimerization is mediated by its B-box domain. These results suggest that the CYLD dimer may interact with the EB1 dimer to modulate microtubule dynamics and to link microtubules to the cell cortex, thereby regulating spindle positioning in mitotic cells. Furthermore, we find that CYLD knockdown results in defects in chromosome segregation and cytokinesis, evidenced by non-horizontal cell division and chromosome lagging. Kinase assays reveal that CYLD negatively regulates the kinase acivity of Aurora-B, while its binding partner EB1 promotes Aurora-B activity. indicating opposite roles for CYLD and EB1 in the regulation of Aurora-B activity. We further find that CYLD knockdown results in the loss of contact-mediated inhibition of cell proliferation, which may be result from abnormal spindle positioning and cytokinesis. These findings suggest that the regulation of spindle positioning, cytokinesis, and Aurora-B activity by CYLD may contribute to its tumor-suppressing function.In conclusion, this study provides the first evidence that CYLD is a microtubule-binding protein and regulates microtubule dynamics, thereby mediating cell migration. This study further demonstrates that CYLD regulates angiogenesis through its effects on the polarization and migration of vascular endothelial cells. In addition, the present study reveals that CYLD regulates spindle positioning by mediating the interaction of astral microtubules with the cell cortex and that CYLD participates in the regulation of chromosome segregation, cytokinesis, and Aurora-B activity. These results provide novel insights into the functions of CYLD in normal physiologic processes and cancer development.