The Role of the Spectraplakin Short-Stop in Cell Migration

描述(申请人提供)：细胞迁移需要肌动蛋白-微管细胞骨架的动力学以及细胞-基质和细胞-细胞黏附的调节；然而，我们对这些网络是如何协调的知识存在一个根本的空白。这一差距阻碍了对控制细胞运动的机制的全面了解，包括转移。该项目的长期目标是确定在细胞迁移过程中黏附和细胞骨架是如何整合的，重点是果蝇spectraplakin Short-Stop和它的哺乳动物对应物MACF1/ACF7是如何协调这些网络的。斑点蛋白是这种协调的极佳候选者，因为它们可以在物理上使肌动蛋白和微管发生交叉连接，同时在黏附调节中发挥关键作用。细胞迁移是这些网络之间协同关系的结果；因此，研究可能促进这种协作相互作用的分子代表着理解这一过程的下一个关键步骤。我们的中心假设是，协调肌动蛋白-微管交联和黏附的分子在细胞迁移的调节中起着纽带的作用。这一假设是基于申请人的实验室中产生的数据以及文献中存在的越来越多的证据而提出的。这项拟议研究的基本原理是，转移是癌症患者死亡的主要原因。对细胞迁移的更广泛的了解将有助于确定未来的治疗目标，并最终减少与癌症相关的死亡。这一假说将通过两个具体的目标来检验：1)确定SPORT和MACF1如何调节细胞迁移；2)确定调节其活性的生物机制。为了实现第一个目标，我将使用一种新型的可移动的果蝇细胞系和具有高分辨率显微镜的哺乳动物组织培养细胞相结合。为了补充这些研究，将在细胞迁移的体内模型中分析ShoT在细胞迁移中的作用。在第二个目标中，将使用生化和显微分析来确定Shot和MACF1是否经历了分子内构象变化。为此，将使用基于细胞生物学和基于发育生物学的分析来确定这种构象变化的生物学意义。这种方法是创新的，因为它将以互补的方式利用果蝇和哺乳动物的细胞培养模型，以及细胞迁移的体内模型。这项研究具有重要意义，因为它的目的是阐明细胞迁移过程中黏附和细胞骨架动力学的组合影响。这一知识有可能推进细胞运动学领域，并可能发现预防转移的治疗靶点。

DESCRIPTION (provided by applicant): Cell migration requires the dynamics of the actin-microtubule cytoskeleton and the regulation of both cell- matrix and cell-cell adhesion; however, there is a fundamental gap in our knowledge of how these networks are coordinated. This gap prevents a comprehensive understanding of the mechanisms that govern cell movements, including metastasis. The long term goal of this project is to determine how adhesion and the cytoskeleton are integrated during cell migration, focusing on how the Drosophila spectraplakin Short-stop and its mammalian counter-part MACF1/ACF7, model cytolinkers and integrators, coordinate these networks. Spectraplakins are excellent candidates for this coordination as they can physically cross-link actin and microtubules while playing a critical roles in the regulation o adhesion. Cell migration is a result of a synergistic relationship between these networks; therefore investigating molecules that potentially facilitate this cooperative interaction represens the next crucial step in understanding this process. Our central hypothesis is that molecules that coordinate actin-microtubule cross-linking and adhesion function as a nexus in the regulation of cell migration. This hypothesis has been formulated on the basis of data generated in the applicant's laboratory as well as mounting evidence present in the literature. The rationale for the proposed research is that metastasis represents a major cause of mortality in cancer patients. A more extensive understanding of cell migration will lead to the identification of futur therapeutic targets and ultimately a decrease in cancer related deaths. This hypothesis will be tested by two specific aims: 1) Determine how the spectraplakins Shot and MACF1 regulate cell migration; and 2) Determine the biological mechanisms that regulate their activity. To accomplish the first aim I will use a combination novel motile Drosophila cell lines and mammalian tissue culture cells with high resolution microscopy. To complement these studies analysis of Shot's role in cell migration will be carried out in an in vivo model of cell migration border cell migration. In the second aim biochemical and microscopic analysis will be used to determine whether Shot and MACF1 undergo an intramolecular conformational change. For this aim both cell biology based and developmental biology based assays will be used to determine the biological significance of this conformational change. The approach is innovative because it will utilize both cell culture models,Drosophila and mammalian, and an in vivo model of cell migration in a complementary manner. The proposed research is significant as its aim is to elucidate the combinatorial affects of adhesion and cytoskeletal dynamics during cell migration. This knowledge has the potential to advance the field of cell motility, and putatively uncover therapeutic targets for the prevention of metastasis.