Arp2/3 Complex and Osteoclast Bone Resorption

描述(由申请人提供)：这项资助计划概述了在生物医学和牙科科学领域追求学术和研究生涯所需的五年培训计划。这位首席研究员目前正在通过佛罗里达大学医学院攻读生物医学博士学位，并将于2003年夏天通过牙科学院开始一个正畸住院医师项目。这项计划将允许首席研究人员通过在蛋白质表达和纯化、细胞培养、信使核糖核酸抑制、质粒构建、病毒载体使用和细胞转导方面的技术培训，获得病毒学、遗传学、分子细胞生物学和生物化学领域的能力。这项提议的直接目标是1)提高生物素养，2)获得先进技术方面的技能，3)激发批判性思维能力，最终目标是为赫斯特博士在学术界和生物医学研究领域的独立职业生涯做好准备。L.Shannon Holliday博士，正畸学系助理教授，将指导首席研究员进行她的研究。霍利迪博士研究破骨细胞激活的分子和细胞生物学，在这项研究涉及的技术方面拥有专业知识，并在破骨细胞生物学领域发表了大量出版物。密歇根大学健康科学中心的广泛研究社区提供了完成这项建议并允许主要研究人员在研究中获得独立所需的所有必要组件，包括协作和物理组件。被称为破骨细胞的特殊细胞的骨吸收是正常生理所必需的。然而，骨吸收增强会导致严重的疾病，包括骨质疏松症和转移性骨肿瘤。尽管破骨细胞介导的疾病通常没有共同的病因，但在每种情况下，破骨细胞必须形成酸性的细胞外腔来降解骨骼。这个项目的重点是破骨细胞和骨骼之间紧密接触的区域，密封区，它隔离了这个酸性隔间。封闭区的形成是骨吸收不可缺少的环节。正如研究计划中所描述的，关于Arp2/3复合体如何参与密封层形成的新假说已经从首席研究人员的研究中演变而来。本项目旨在验证这一新的假设，并有三个具体的目标：第一，确定在体内形成封闭区所需的Arp2/3；第二，表征Arp2/3调节蛋白Cortactin；第三，表征Arp2/3复合体的调节蛋白血管扩张剂刺激的磷酸蛋白(Vasp)对降钙素的反应，并确定其对肌动蛋白环形成的影响。通过研究Arp2/3复合体在封闭区的结构、调节和功能作用，有可能为基因治疗或传统药物抑制骨吸收疾病寻找新的靶点。

DESCRIPTION (provided by applicant): This grant proposal outlines a five-year training program required for the pursuit of an academic and research career in biomedical and dental sciences. The principal investigator is currently pursuing a PhD in Biomedical Sciences through the University of Florida College of Medicine and concomitantly will be beginning an orthodontic residency program through the College of Dentistry in the summer of 2003. This program will allow the principal investigator to gain competence in the areas of virology, genetics, molecular cell biology and biochemistry, by technical training in protein expression and purification, cell culture, mRNA inhibition, plasmid construction, viral vector use, and cell transduction. The immediate goals of this proposal are to 1) increase biological literacy, 2) gain skills in advanced techniques, and 3) stimulate critical thinking skills, with the ultimate goal being the preparation of Dr. Hurst for an independent career in academia and biomedical research. Dr. L. Shannon Holliday, PhD, an Assistant Professor in the Department of Orthodontics, will mentor the principal investigator in her research. Dr. Holliday studies the molecular and cellular biology of osteoclast activation, has expertise in the techniques involved in this study, and has numerous publications in the field of osteoclast biology. The extensive research community at the UF Health Science Center provides all of the necessary components, collaborative and physical, required to complete this proposal and allow the principal investigator to gain independence in research. Bone resorption by specialized cells called osteoclasts is required for normal physiology. However, enhanced bone resorption can cause severe disease, including osteoporosis and metastatic bone tumors. Although osteoclast-mediated diseases often do not share common etiologies, in each case, osteoclasts must form an acidic extracellular compartment to degrade bone. This project focuses on a region of tight contact between the osteoclast and bone, the sealing zone, which segregates this acidic compartment. Sealing zone formation is indispensable for bone resorption. As described in the research plan, a new hypothesis for how the Arp2/3 complex is involved in sealing zone formation has evolved from the principal investigator's studies. This project is designed to test this novel hypothesis and has three specific aims: first, to determine the requirement of Arp2/3 for the formation of the sealing zone in vivo; second, to characterize the Arp2/3 regulatory protein, cortactin; third, to characterize phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a regulatory protein of the Arp2/3 complex, in response to calcitonin and determine its effects on actin ring formation. By studying the structural, regulatory and functional roles of the Arp2/3 complex in the sealing zone, identification of new targets for gene therapy or conventional pharmaceuticals to inhibit resorptive bone diseases may be possible.