Amyloids, aggregates and Nucleolar activity in Xenopus development

项目摘要： 许多对正常发育至关重要的细胞过程，当它们被破坏时， 不受控制。不久前，蛋白质聚集在很大程度上等同于发病机制。更 最近，我们知道蛋白质聚集介导应激反应，细胞分裂，长期 记忆，并在细胞核和胞质溶胶中自组装成关键的非膜结合颗粒。如果 事实上，至少5%的已知蛋白质具有允许它们形成聚集体的组成。 缺乏对蛋白质聚集的适当细胞控制导致淀粉样蛋白形成，其不受调节， 阿尔茨海默氏症和亨廷顿舞蹈症等毁灭性疾病的发病机制 疾病和常见的使人衰弱的疾病，如白内障。骨料的物理性质可能是 相分离的液滴、凝胶、无序聚集体和有序聚集体 淀粉样蛋白的特征。聚集体的作用是大大增加局部的浓度， 聚集蛋白质，并且是不同结构状态之间平衡的一部分。 因此，蛋白质加入或离开聚集体的能力和速率是一个重要特征 这是可以调节的，如果受到损害，就会导致基于聚集的病理学。有差距 了解哪些蛋白质参与聚集体的形成，以及聚集体是如何被调节的。 vivo.我们认为，开始弥合差距的理想系统是青蛙的卵母细胞 非洲爪蟾我们和其他人已经表明，聚集体在卵子发生过程中积累， 细胞质和细胞核中。核颗粒负责RNA转录和加工的所有 三种RNA聚合酶通常与作为共聚集体的RNA自组装成聚集体。的 这个建议的重点是核仁，一个突出的非膜结合颗粒负责 核糖体RNA的合成和加工，核糖体的制造，帮助介导 细胞对压力和DNA损伤的反应，并适当地响应细胞对新物质的需求 翻译能力。我们使用显微镜，免疫组织化学，生化分离，蛋白质 消耗，蛋白质组学和转录组学分析，以了解积累，功能和稳定性 形成核仁的形式和功能。我们利用了 非洲爪蟾卵母细胞核的性质，包括大小，丰富的蛋白质和RNA含量和容易 操纵来完成我们的研究这些发现将影响我们对细胞如何维持 天然和聚集形式的蛋白质之间的生理正常平衡。

Project Summary: Many cellular processes critical for normal development have devastating consequences when they are not controlled. Not too long ago, protein aggregation was largely equated with pathogenesis. More recently we know that proteins aggregate to mediate stress responsiveness, cell division, long-term memory and to self-assemble into critical non-membrane bound particles in the nucleus and cytosol. If fact, at least 5% of known proteins have the composition that would allow them to form aggregates. Lack of proper cellular control of protein aggregation leads to amyloid formation, which unregulated, is part of the pathogenesis that accompanies devastating diseases like Alzheimer’s and Huntington’s disease and common debilitating conditions like cataracts. The physical nature of aggregates may be that of phase separated liquid droplets, gels, disordered aggregates and the ordered aggregates characteristic of amyloid. Aggregates serve to greatly increase the local concentration of the aggregating protein, and are one part of an equilibrium between different structural states. Consequently, the ability and rate of proteins joining or leaving an aggregate is an important feature that can be regulated, and if compromised leads to aggregate based pathology. There is a gap in understanding which proteins participate in aggregate formation and how aggregation is regulated in vivo. We propose that an ideal system to begin to bridge the gap would be the oocytes of the frog Xenopus laevis. We and others have shown that aggregates accumulate during oogenesis in the cytosol and in nucleus. The nuclear particles responsible for RNA transcription and processing by all three RNA polymerases self-assemble into aggregates, generally with RNA as a co-aggregate. The focus of this proposal is on nucleolus, a prominent non-membrane bound particle responsible for synthesis and processing of ribosomal RNA, the manufacturing of ribosomes, helping to mediate cellular responses to stress and DNA damage, and appropriately responding to cellular need for new translational capacity. We use microscopy, immunohistochemistry, biochemical fractionation, protein depletion, proteomic and transcriptomic analysis to understand the accumulation, function and stability of the aggregates that establish nucleolar form and function. We take advantage of the unique properties of Xenopus oocyte nuclei, including size, abundant protein and RNA content and ease of manipulation to perform our studies. The findings will impact our understanding of how cells maintain physiologically normal equilibrium between native and aggregating forms of proteins.