The Role of the Spectraplakin Short-Stop in Cell Migration

描述（由申请人提供）：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-细胞粘附的调节；然而，我们对这些网络如何协调的认识存在根本性的差距。这一差距阻碍了对控制细胞运动（包括转移）的机制的全面理解。该项目的长期目标是确定在细胞迁移过程中粘附和细胞骨架是如何整合的，重点关注果蝇谱板Short-stop及其哺乳动物对应部分MACF1/ACF7、模型细胞连接子和整合子如何协调这些网络。光谱蛋白是这种协调的优秀候选者，因为它们可以物理交联肌动蛋白和微管，同时在粘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，研究可能促进这种合作相互作用的分子是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-微管交联和粘附的分子在细胞迁移调节中起着联系作用。这一假设是根据申请人实验室产生的数据以及文献中出现的越来越多的证据制定的。提出这项研究的基本原理是，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导致确定未来的治疗靶点，并最终减少癌症相关的死亡。这一假设将通过两个特定目的进行验证：1)确定谱蛋白Shot和MACF1如何调节细胞迁移；2)确定调节其活性的生物学机制。为了实现第一个目标，我将使用一种新型的活动果蝇细胞系和哺乳动物组织培养细胞的高分辨率显微镜。为了补充这些研究，Shot在细胞迁移中的作用将在细胞迁移边界细胞迁移的体内模型中进行。在第二个目标中，生化和显微分析将用于确定Shot和MACF1是否经历分子内构象变化。为此目的，基于细胞生物学和基于发育生物学的分析将被用来确定这种构象变化的生物学意义。这种方法是创新的，因为它将利用细胞培养模型，果蝇和哺乳动物，以及细胞迁移的体内模型，以互补的方式。本研究的目的是阐明细胞迁移过程中粘附和细胞骨架动力学的综合影响。这一知识有可能推动细胞运动领域的发展，并推测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.