【作者】 高金珉；

【导师】 周军；

【作者基本信息】 南开大学 ， 遗传学， 2010， 博士

【摘要】 微管细胞骨架在多种细胞活动过程中发挥着非常重要的作用,如细胞形态的维持、胞内物质运输、细胞分裂以及细胞运动。微管具有动态性,能不断进行聚合与解聚的动态变化,这种动态性对于其很多功能的发挥至关重要。细胞内微管的功能及动态性是由一系列的微管结合蛋白调节的,包括调节微管组装、组织的蛋白以及介导细胞器和膜泡运输的马达蛋白。微管结合蛋白的异常表达或药物处理会使细胞内微管的组装或稳定性发生异常,进而可能导致严重的细胞表型,如细胞运动的缺陷、细胞周期停滞甚至细胞死亡。CYLD是一个由956个氨基酸组成的蛋白质,其基因的突变与人类家族性圆柱瘤等肿瘤的发生有着密切的关系。CYLD的羧基端含有一个去泛素化酶催化结构域,赋予该蛋白去泛素化酶的功能,能特异性去除靶蛋白上的通过第63位赖氨酸连接形成的泛素链。另外,CYLD的氨基端含有三个CAP-Gly结构域。CAP-Gly结构域存在于多种微管结合蛋白中,并介导这些蛋白与微管的相互作用。然而,尚不清楚CYLD是否能够与微管相互作用,如果能够相互作用,是否是通过其CAP-Gly结构域来介导。在本项研究中,我们通过免疫荧光染色实验,发现在细胞中内源的CYLD与微管共定位。微管共沉淀实验表明CYLD能够直接与微管在体外相互作用。微管共沉淀实验进一步显示,CYLD含有三个CAP-Gly结构域的氨基端以及其第一个CAP-Gly结构域主要存在于微管沉淀中,表明CYLD与微管的相互作用主要由CYLD的第一个CAP-Gly结构域来介导。通过GST下拉实验,我们发现CYLD也能够与微管的亚基——微管蛋白相互作用。细胞内CYLD表达的沉默能够显著抑制微管解聚后的再生,表明CYLD具有促进微管聚合的功能。通过微管蛋白混浊度检测实验,我们发现CYLD能够在体外促进微管蛋白聚合成微管,并且在稀释诱导的微管解聚过程中对微管具有稳定作用。而且,微管蛋白混浊度检测实验以及微管沉淀实验都证实CYLD是通过降低微管聚合的临界浓度而促进微管蛋白聚合成微管。另外,通过划痕愈合实验,我们发现CYLD在介导细胞迁移的过程中发挥着重要的作用,并且CYLD的这一调节活性依赖于其第一个CAP-Gly结构域。这些研究结果表明CYLD是一个对微管动态性具有重要调节作用的CAP-Gly蛋白。虽然CYLD最初是作为一个肿瘤抑制因子被发现的,但它最近被发现在多种重要的生理活动中起作用,如免疫应答、炎症反应、破骨细胞发生、细胞周期调控等。在本项研究中,我们发现CYLD在血管新生中也发挥着非常重要的作用。血管新生是一个受到促血管新生因子和抑血管新生因子调制的过程,并依赖于血管内皮细胞的迁移。在基于基质胶(matrigel)的内皮细胞成管实验和基于胶原胶的细胞球出芽形成实验中,我们发现了CYLD表达的沉默使得血管新生的能力显著削弱。我们进一步研究了CYLD对体内血管新生的影响,发现CYLD的小干扰RNA及CYLD的抗体在小鼠体内能显著抑制血管新生诱导因子诱导的血管生成。我们接下来研究了CYLD调节血管新生的分子机制。细胞铺展实验和划痕愈合实验显示CYLD通过介导血管内皮细胞的铺展和迁移来调节血管新生。我们对迁移中细胞的前沿膜皱褶的动态性进行了检测,发现CYLD表达抑制的细胞中膜皱褶的动态性显著降低,进一步证明了CYLD对血管内皮细胞迁移的调节。CYLD表达的沉默显著抑制了内皮细胞中微管的动态性,并通过阻止细胞的极化而抑制内皮细胞的迁移,这些结果表明CYLD对微管动态性的调节在细胞迁移中发挥重要作用。为了进一步地了解CYLD调节内皮细胞迁移和血管新生的机制,我们研究了CYLD的调节作用是否涉及到了Rho家族GTP酶。结果表明,Rac1的活化在CYLD调节血管内皮细胞迁移和血管新生过程中起着重要的作用。这些研究结果揭示CYLD具有调节血管新生的功能,并提出在内皮细胞迁移和血管新生过程中Rac1活化的一种新机制。CYLD已经被发现在细胞周期进程中具有重要的调节作用,然而其详细的分子机制仍需进一步地了解。在细胞进入有丝分裂的时候,微管经历剧烈的重组过程。在这个过程中,精确的微管动态性对于有丝分裂纺锤体的组装以及染色体的分离是必需的。我们的研究发现CYLD调节微管的动态性,这暗示了CYLD可能在有丝分裂进程中具有重要作用。为了检测这种可能性,我们研究了CYLD对纺锤体组装的影响,发现CYLD表达的降低能够导致纺锤体的异常定位,表明CYLD对于有丝分裂纺锤体的定位具有关键作用。我们通过免疫荧光染色实验,发现CYLD介导了星体微管与细胞皮层的相互作用。微管末端结合蛋白EB1已经被发现能够介导微管正极与细胞皮层之间的连接。我们通过GST下拉实验,发现CYLD能够与EB1相互作用,并通过免疫荧光染色实验,发现CYLD与EB1在部分星体微管的末端存在共定位。通过GST下拉实验,我们进一步发现CYLD在细胞内能够形成二聚体,这种二聚化作用是由CYLD的B-box结构域介导的。这些结果暗示CYLD二聚体可能与EB1二聚体相互作用来调节微管的动态性和介导微管对细胞皮层的捕获,进而调节有丝分裂细胞中纺锤体的定位。另外,我们的研究发现CYLD表达的降低能诱导染色体分离及胞质分裂异常,表现为细胞在非水平的方向进行分裂和存在染色体分离滞后的现象。激酶实验显示CYLD能够负调节Aurora-B激酶的活性,而CYLD的结合蛋白EB1则促进Aurora-B激酶的活性,这些结果暗示CYLD和EB1对Aurora-B激酶的活性调节具有相反的作用,共同调节胞质分裂进程。我们的研究进一步地表明CYLD表达水平的降低导致细胞增殖不受接触抑制的控制,而细胞增殖接触抑制的丧失可能是由纺锤体定位和胞质分裂的异常所引起的。我们的研究表明,CYLD对纺锤体定位、胞质分裂以及Aurora-B活性的调节可能是其发挥肿瘤抑制功能的重要因素。概括来讲,本项研究首次证明CYLD是一个微管结合蛋白,能够调节微管的动态性,从而在细胞迁移过程中发挥重要作用。本项研究进一步发现CYLD能够通过影响血管内皮细胞的极化和迁移而调节血管的新生。此外,本项研究还发现CYLD能够通过介导星体微管与细胞皮层的相互作用而影响有丝分裂纺锤体的定位,并参与对于染色体分离、胞质分裂以及Aurora-B活性的调节。这些研究结果为理解CYLD在正常生理活动及肿瘤发生过程中的作用提供了崭新的思路。更多还原

【Abstract】 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.更多还原