The Role of the Spectraplakin Short-Stop in Cell Migration

Spectraplakin 短停在细胞迁移中的作用

基本信息

批准号：
8850562
负责人：
Derek Anthony Applewhite
金额：
$ 8.73万
依托单位：
REED COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8850562
关键词：
Actins Address Adhesions Affect Behavior Biochemical Biochemistry Biological Biological Assay Cancer Patient Cell Adhesion Cell Culture Techniques Cell Line Cell Polarity Cell-Cell Adhesion Cell-Matrix Junction Cells Cellular biology Cessation of life Complement Core Facility Critiques Cytoplasm Cytoskeleton Data Databases Development Developmental Biology Disease Disseminated Malignant Neoplasm Drosophila genus Embryo Embryonic Development Environment Epithelium Equipment Event Family Family member Feedback Future Gap Junctions Genetic Goals Hemocytes In Vitro Individual Invaded Knowledge Lead Literature Maintenance Malignant Neoplasms Mentors Microscope Microscopic Microscopy Microtubules Modeling Molecular Molecular Motors Neoplasm Metastasis Organ Physiological Play Process Protein Family Proteins RNA Interference Regulation Research Research Personnel Resolution Resources Role System Systems Analysis Testing Tissues Traction Training Work Writing base cell motility combinatorial crosslink fly in vivo in vivo Model innovation insight interest meetings member metastasis prevention metastatic process migration mortality novel post-doctoral training protein protein interaction public health relevance skills synergism therapeutic target tissue culture tissue/cell culture

项目摘要

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.

描述（由申请人提供）：我有兴趣了解细胞迁移和转移。我的博士后训练集中在S2细胞中的模型细胞链接和积分器的果蝇Spectraplakin短路（SHOT）的细胞生物学上。我已经确定了将射击功能与肌动蛋白细胞骨架交联的链接微管，并且这样做的是分子电机的力，否则它们会在细胞质中通过鞭打并扣为游离微管。镜头不仅是交联的肌动蛋白和微管，而且是EB1依赖性和终端跟踪蛋白（+TIP）。我已经确定了哪些残留物用于与EB1的相互作用。继续这个项目，我将确定射击如何调节细胞迁移并确定镜头自身法规背后的机制。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自己的技能，以采取一种全面的方法来研究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 - 马trix和细胞细胞粘附的调节，但是，我们了解这些净作品如何协调的基本差距。持续存在这一差距代表了一个重要的问题，因为在填补之前，对控制转移的机制的全面理解将仍然难以捉摸。长期目标是确定在细胞迁移过程中如何整合粘附和细胞骨架。该应用程序的目的是确定在细胞迁移过程中射击如何协调这些网络的方式。 Shot是这种协调的绝佳候选者，因为它可以在细胞 - 矩阵和细胞细胞粘附的调节和维持中扮演对果蝇胚胎的发展至关重要的角色时进行物理交联的肌动蛋白和微管。细胞迁移是这些网络之间协同关系的结果，研究了可能促进这种协同作用的分子，代表了理解这一过程的下一个关键步骤。我们的中心假设是，在调节细胞迁移的调节中，协调肌动蛋白 - 微管交联和粘附功能的分子。该假设是根据我在我生成的数据以及文献中存在的越来越多的证据的基础上提出的。拟议的搜查的理由是转移代表了癌症患者死亡率的主要原因。细胞迁移的全面理解将导致未来的治疗靶标和癌症相关死亡的减少。该假设将通过两个具体的目的来检验：1）确定射击如何机械调节细胞迁移； 2）确定镜头的调节机制。为了实现第一个目标，将使用一种新型的果蝇上皮细胞系，可与RNAi合作，并使用高分辨率显微镜。该细胞系在组织培养物中很容易形成细胞矩阵和细胞 - 细胞粘附，这方面将被大写以研究Shot在两者的形成和调节中的作用。为了补充这些研究，对SHOT在细胞迁移中的作用的分析将在两个细胞迁移，边界细胞迁移和血细胞迁移的体内模型中进行。在第二个目标中，将使用生化和微观分析来确定射击是否发生分子内构象变化，并且基于细胞生物学和基于发育生物学的测定法将用于阐明这种构象变化的生物学意义。这种方法具有创新性，因为它以互补的方式利用细胞迁移的细胞培养模型和果蝇胚胎发生。拟议的研究很重要，因为预计它将阐明细胞迁移过程中粘附和细胞骨架动力学的组合影响。这些知识有潜力推进细胞运动领域，并推动了预防转移的治疗靶标。 UNC的研究环境非常支持和协作。除了Rogers博士的持续培训和指导外，我还将与Droso-Phila遗传学和发展生物学专家Mark Peifer博士紧密合作，而Kevin Slep博士则是一位成就良好的结晶学家和生物化学家，以获得我的培训目标。在Rogers，Peifer和SLEP实验室的资源之外，我可以通过核心设施和部门设施的组合来访问许多尖端显微镜和设备。此外，UNC有许多我可以寻求专业知识的调查员，并且通过校园广泛的研讨会，我将有机会定期与他们互动并获得重要的反馈。