SGER: Exploration of a Novel Nuclear Domain Tightly Associated with PML Bodies

这是探索性研究的一小笔助学金。劳伦斯将寻求最近的初步证据，证明有一个新的核“区域”或“身体”，它通常位于紧邻PML小体的位置，并似乎与PML小体形成“双重体”结构。近年来，人们越来越多地认识到内部核结构的复杂性和重要性，越来越多的证据表明它们在各种核功能中发挥着基础性作用。最前沿的是对PML小体(也称为ND10、Pod或PML结构域)的浓厚兴趣，因为这些结构丰富了一系列重要的基因调控蛋白。劳伦斯博士的实验室一直在研究不同核区的特征，以及它们与基因表达的结构和功能关系。在她最近的工作过程中，她偶然发现了证据，表明存在一个在结构上与PML机构有联系的基本上未知和未刻画特征的核“机构”。关于这一点的证据还处于非常初步的状态，还有一些重大问题需要解决，以便最终确定这是一个包含多种核因素并具有明确结构的真正核机构的存在。这项工作的潜在意义在于，它可能展示细胞结构中一个以前未知的部分，相当于一个在基本核功能中具有未知角色的核“细胞器”。此外，这些小体在结构上与一个明显与细胞周期调节有关的小体相连。劳伦斯博士和她的同事最初使用针对标志表位的抗体在细胞中发现了一个由10-20个非常明亮的核“点”(紧邻但不重叠)组成的规则间隔的图案，这些细胞被设计成在生理水平上表达标志标记的c-myc癌蛋白。然而，最近的证据表明，检测到的抗原可能不是myc癌蛋白，而是一种内源性蛋白，可能是作为对MuLV病毒(小鼠白血病病毒)的细胞反应的一部分而诱导的。第一个目标是利用超微结构分析和免疫荧光对先前证据表明可能共同定位于这些小体的其他几个特定蛋白质进行研究，以调查PML相关小体确实存在并在各种细胞中被发现的假设。其次，她将研究MuLV载体或MuLV表达与相关蛋白和/或体的诱导之间的关系。根据结果，通过抗FLAG抗体检测并定位于这些结构的未知细胞蛋白也是可行的。

This is a Small Grant for Exploratory Research.Dr. Lawrence will pursue recent preliminary evidence of a novel nuclear "domain" or "body," which typically positions immediately adjacent to PML bodies and appears to form a "doublet" structure with the PML body. In recent years, there has been increasing appreciation for the complexity and importance of internal nuclear structures and growing evidence of their fundamental roles in diverse nuclear functions. At the forefront has been intense interest in PML bodies (also called ND10, PODs, or PML domains), because these structures are enriched for a collection of important gene regulatory proteins. Dr. Lawrence's laboratory has been studying the characterization of distinct nuclear compartments and their structural and functional relationship to gene expression. In the course of her very recent work she fortuitously uncovered evidence that there exists an essentially unknown and uncharacterized nuclear "body" that is structurally linked to PML bodies. The evidence for this is at a very preliminary state, and there are major questions to be sorted out in order to establish conclusively the existence of this as a bona fide nuclear body containing multiple nuclear factors and having defined structure. The potential significance of the work is that it may demonstrate a previously unknown part of cell structure, the equivalent of a nuclear "organelle" with an unexplored role in basic nuclear function. In addition, these bodies are structurally coupled with a body that is clearly implicated in cell cycle regulation. Dr. Lawrence and her colleagues initially discovered a regularly spaced pattern of ~10-20 very bright nuclear "dots" (immediately abutting but not overlapping PML bodies) using an antibody to the FLAG-epitope in cells engineered to express flag-tagged c-myc oncoprotein at physiological levels. However, recent evidence suggests that the antigen detected is probably not the myc oncoprotein but rather an endogenous protein, possibly induced as part of a cellular reaction to MuLV virus (murine leukemia virus). The first aim is to investigate the hypothesis that PML-associated bodies indeed exist and are found in a variety of cells, using both ultrastructural analysis and immunofluorescence to several other specific proteins that prior evidence suggests may co-localize to these bodies. Second, she will investigate the relationship between MuLV vectors or MuLV expression and the induction of the relevant protein and/or body. Depending on results, it would also be feasible to identify an unknown cellular protein detected by the anti-flag antibodies and localized to these structures.