The Role of the Spectraplakin Short-Stop in Cell Migration

描述(申请人提供)：我对了解细胞迁移和转移感兴趣。我的博士后培训主要集中在果蝇spectraplakin Short-Stop(Sort)的细胞生物学上，它是S2细胞中的一种细胞连接和整合模型。我已经确定Shot的功能是将微管与肌动蛋白细胞骨架交联，在这样做的过程中，与分子马达的力量相反，分子马达会在细胞质中鞭打和弯曲自由的微管。Shot不仅是肌动蛋白和微管的交联体，而且是一种依赖EB1的正端跟踪蛋白(+Tip)。我已经确定了拍摄的残基用于与EB1的相互作用。继续这个项目，我将确定Shot如何调节细胞迁移，并确定Sort自身调节背后的机制。我将通过结合果蝇遗传学和发育生物学以及生物化学来扩展我的技能，以便采取全面的方法来研究Shot在细胞迁移中的作用。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认识存在着一个根本的空白。这一缺口的持续存在是一个重要的问题，因为在填补这一缺口之前，对转移机制的全面了解仍将是难以捉摸的。长期目标是确定黏附和细胞骨架在细胞迁移过程中是如何整合的。这个应用程序的目标是确定在细胞迁移过程中Shot如何协调这些网络。Sort是这种协调的极佳候选者，因为它可以物理上连接肌动蛋白和微管，同时在调节和维持细胞-基质和细胞-细胞黏附方面发挥对果蝇胚胎发育至关重要的作用。细胞迁移是这些网络之间协同关系的结果，研究可能促进这种协同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。这一假设是基于我在中生成的数据以及文献中提供的越来越多的证据而提出的。提出这项研究的理由是，转移是癌症患者死亡的主要原因。对细胞迁移的全面了解将导致未来的治疗目标和癌症相关死亡的减少。这一假说将通过两个具体的目标来检验：1)确定Shot如何机械地调节细胞迁移；2)确定Shot的调节机制。为了实现第一个目标，将使用一种新的可移动的果蝇上皮细胞系，该细胞系符合RNAi和高分辨率显微镜。这种细胞系在组织培养中很容易形成细胞基质和细胞间的黏附，这一方面将被用来研究Shot在两者的形成和调节中的作用。为了补充这些研究，将在细胞迁移、边界细胞迁移和血细胞迁移两种体内模型中分析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是否经历了分子内构象变化，并将使用基于细胞生物学和发育生物学的分析来阐明这种构象变化的生物学意义。这种方法是创新的，因为它以互补的方式利用了细胞迁移和果蝇胚胎发生的细胞培养模型。这项拟议的研究具有重要意义，因为它有望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。北卡罗来纳大学的研究环境一直非常支持和合作。除了罗杰斯博士的持续培训和指导外，我还将与马克·皮弗博士(Droso-Phila遗传学和发育生物学专家)以及凯文·斯莱普博士(Kevin SLEP博士)密切合作，以实现我的培训目标。除了罗杰斯、皮弗和SLEP实验室的资源外，我还可以通过核心设施和部门设施的组合来获得一些尖端显微镜和设备。此外，北卡罗来纳大学有许多我可以寻求的具有专业知识的调查人员，通过校园研讨会，我将有机会定期与他们互动并收到关键的反馈。

DESCRIPTION (provided by applicant): I am interested in understanding cell migration and metastasis. My postdoctoral training has focused on the cell biology of the Drosophila spectraplakin Short-stop (Shot), a model cytolinker and integrator, in S2 cells. I have determined that Shot functions to cross-link microtubules to the actin cytoskeleton, and in doing so opposes the forces of molecular motors which would otherwise whip and buckle the free microtubules through- out in the cytoplasm. Shot not only cross-links actin and microtubules, but is also an EB1-dependent plus end tracking protein (+Tip). I have determined which residues Shot uses for its interaction with EB1. Continuing this project, I will determine how Shot regulates cell migration and determine the mechanism behind Shot's own regulation. I will expand upon my skill set by incorporating Drosophila genetics and developmental biology, and biochemistry in order to take a comprehensive approach to investigating Shot's role in cell migration. 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 net- works are coordinated. Continued existence of this gap represents an important problem because until it is filled, a comprehensive understanding of the mechanisms that govern metastasis will remain elusive. The long term goal is to determine how adhesion and the cytoskeleton are integrated during cell migration. The objective of this application is to identify how the Shot coordinates these networks during cell migration. Shot is an excellent candidate for this coordination as it can physically cross-link actin and microtubules while playing roles critical to the development of the Drosophila embryo in the regulation and maintenance of both cell-matrix and cell-cell adhesion. Cell migration is a result of a synergistic relationship between these networks, investigating molecules that potentially facilitate this synergism represents 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 I generated in as well as mounting evidence present in the literature. The rationale for the proposed re- search is that metastasis represents a major cause of mortality in cancer patients. A comprehensive under- standing of cell migration will lead to future therapeutic targets and a decrease in cancer related deaths. This hypothesis will be tested by two specific aims: 1) Determine how Shot mechanistically regulates cell migration; and 2) Determine Shot's regulatory mechanism. To accomplish the first aim, a novel motile Drosophila epithelia cell line amenable to RNAi and high resolution microscopy will be used. This cell line readily forms cell-matrix and cell-cell adhesion in tissue culture and this aspect will be capitalized upon to study Shot's role in the formation and regulation of both. To complement these studies, analysis of Shot's role in cell migration will be carried out in two in vivo models of cell migration, border cell migration and hemocyte migration. In the second aim biochemical and microscopic analysis will be used to determine whether Shot undergoes an intramolecular conformational change, and both cell biology based and developmental biology based assays will be used to elucidate the biological significance of this conformational change. The approach is innovative because it utilizes both cell culture models of cell migration and Drosophila embryogenesis in a complementary manner. The proposed research is significant because it is expected 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. The research environment at UNC has been extremely supportive and collaborative. In addition to the continued training and mentoring from Dr. Rogers, I will work closely with Dr. Mark Peifer an expert in Droso- phila genetics and developmental biology and Dr. Kevin Slep an accomplished crystallographer and biochemists to obtain my training goals. Outside of the resources Rogers, Peifer, and Slep labs, I have access to a number of cutting edge microscopes and equipment through a combination of core facilities and departmental facilities. Furthermore, UNC has a number of investigators with expertise I can seek and through campus wide seminars I will have the opportunity to regularly interact with them and receive crucial feedback.