Reversible disassembly of the nucleolus by FRGY proteins

核仁是一个动态细胞器。它高度有序的结构被完全分解并准确地 在高等真核生物有丝分裂期间重组。在间期细胞中，核仁蛋白持续而迅速地 穿梭于核仁和核质之间。然而，这些核仁背后的分子机制 动态定义不明确。这项研究的长期目标是确定组装和拆卸的分子基础 核仁组织。研究人员最近发现，非洲爪哇生殖细胞蛋白FRGY2a和FRGY2a FRGY2b可在体外和体内可逆性地解离体细胞核仁。它们是第一批具有这种能力的蛋白质。 从生理上讲，FRGY2a/b和人类同系物YB1是有丝分裂核仁分解所必需的蛋白质 活体细胞中核仁和核质之间的穿梭与癌症引起的核仁解体 化疗药物。为进一步研究FRGY2a/b和YBI对核仁的拆解，提出了三个具体的目标。 (SA1)建立FRGY2a/b和YBI对核仁分解的生理作用。核仁解体活性和 YB1复合体在化学核仁分解、有丝分裂核仁分解的背景下的磷酸化 核仁蛋白质穿梭将使用人类体细胞进行研究。解体核仁的早期维持 将通过显微注射FRGY2a/b的显性负突变体来研究非洲爪哇胚胎。(SA2)了解 YB1解体核仁的分子机制。核仁解体过程与亚核仁定位 将用免疫荧光显微镜和电子显微镜对YB1进行研究。与YB1相互作用的蛋白质 核仁拆解过程中，通过亲和纯化将其分离出来。这些蛋白质的功能将通过 鉴定亚核定位、上调(通过转染法)和下调(通过短干扰RNA和 细胞内蛋白质的反义)。YB1磷酸化对与相互作用蛋白结合的影响将是 也进行了研究。(SA3)鉴定卵母细胞提取液中FRGY2a/b的抑制物S。非洲爪哇的卵被称为 卵母细胞直到排卵，与卵子不同，它们有多个核仁。卵母细胞含有FRGY2a/b及其抑制物(S)。 将采用免疫亲和纯化、His-tag下拉和常规柱纯化的方法分离出 抑制剂。抑制物在间期细胞中的作用，强调核仁的维持和重组 末期的核仁将通过分析其表达模式、上调和下调来研究。这些项目 是重要的，因为最近的研究表明，核仁的功能不仅限于核糖体的合成，而且 涵盖更广泛的细胞生物学领域，如细胞周期控制、癌细胞增殖和端粒酶 监管。拟议的研究结果将极大地有助于理解核仁。 动态，使我们能够调节其不同的功能，以实现医疗利益。

The nucleolus is a dynamic organelle. Its highly organized structure is completely disassembled and accurately reassembled during mitosis in higher eukaryotes. In interphase cells, nucleolar proteins are constantly and rapidly shuttling between the nucleolus and nucleoplasm. However, molecular mechanisms underlying these nucleolar dynamics are ill defined. The long-term goal of this research is to identify molecular basis of assembly and disassembly of the nucleolar organization. The investigators have recently found that Xenopus germ cell proteins FRGY2a and FRGY2b can reversibly disassemble somatic nucleoli in vitro and in vivo. They are the first proteins with this capability. Physiologically, FRGY2a/b and the human homologue YB1 are essential for mitotic nucleolar disassembly, protein shuttling between nucleoli and nucleoplasm in living somatic cells and nucleolar disassembly induced by a cancer chemotherapy drug. Three specific aims are proposed to further study nucleolar disassembly by FRGY2a/b and YBI. (SA1) Establish physiological roles of nucleolar disassembly by FRGY2a/b and YBI. Nucleolar disassembly activity and phosphorylation of the YB1 complex in the context of chemical nucleolar disassembly, mitotic nucleolar disassembly and nucleolar protein shuttling will be studied using human somatic cells. Maintenance of disassembled nucleoli in early Xenopus embryos will be studied by microinjection of dominant negative mutants of FRGY2a/b. (SA2) Understand the molecular mechanisms of nucleolar disassembly by YB1. Nucleolar disassembly process and subnucleolar localization of YB1 will be studied with immunofluorescence microscopy and electron microscopy. Proteins interacting with YB1 during nucleolar disassembly will be isolated by affinity purification. Functions of these proteins will be studied through identification of subnuclear localization, up- (by transfection)and down-regulation (by short interfering RNA and antisense) of the proteins within cells. Effects of YB1 phosphorylation on the binding to the interacting proteins will be also studied. (SA3) Identify and characterize inhibitor(s) of FRGY2a/b in oocyte extract. Xenopus eggs are called oocytes until ovulation and they have multiple nucleoli unlike eggs. Oocytes contain FRGY2a/b and its inhibitor(s). Immunoaffinity purification, His-tag pull-down and conventional column purification will be employed to isolate the inhibitor. Roles of the inhibitor with the emphasis on maintenance of nucleoli in interphase cells and reassembly of nucleoli in telophase will be investigated by analysis of its expression pattern, up- and down-regulation. These projects are important because recent studies show that the function of the nucleolus is not limited to ribosome synthesis but encompasses more wide areas in cell biology such as cell cycle control, cancer cell proliferation and telomerase regulation. The outcome of the proposed research will significantly contribute to the understanding of the nucleolar dynamics, enabling us to regulate its diverse functions for medical benefits.