Arp2/3 Complex and Osteoclast Bone Resorption

描述（由申请人提供）：此拨款提案概述了在生物医学和牙科科学领域从事学术和研究工作所需的五年培训计划。首席研究员目前正在佛罗里达大学医学院攻读生物医学博士学位，并将于2003年夏天开始在牙科学院进行正畸住院医师培训。通过蛋白质表达和纯化、细胞培养、mRNA抑制、质粒构建、病毒载体使用和细胞转导等方面的技术培训，该项目将使首席研究员获得病毒学、遗传学、分子细胞生物学和生物化学领域的能力。该提案的直接目标是1)提高生物素养，2)获得先进技术技能，3)激发批判性思维技能，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。正畸系助理教授L. Shannon Holliday博士将指导首席研究员的研究。Holliday博士研究破骨细胞活化的分子和细胞生物学，在这项研究中涉及的技术方面具有专业知识，并在破骨细胞生物学领域发表了大量论文。在UF健康科学中心广泛的研究社区提供了所有必要的组成部分，协作和物理，需要完成这一提案，并允许主要研究者在研究中获得独立性。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，骨吸收增强可引起严重的疾病，包括骨质疏松症和转移性骨肿瘤。虽然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细胞外腔室来降解骨骼。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，它隔离了酸性隔间。封闭带的形成对于骨吸收是必不可少的。正如研究计划中所描述的那样，首席研究员的研究发展出了一个关于Arp2/3复合体如何参与密封带形成的新假设。本项目旨在验证这一新颖的假设，并有三个具体目标：首先，确定体内密封区形成所需的Arp2/3；二是表征Arp2/3调控蛋白的结构；第三，表征血管扩张剂刺激磷酸化蛋白（VASP）的磷酸化，VASP是Arp2/3复合物的调节蛋白，响应降钙素并确定其对肌动蛋白环形成的影响。通过研究Arp2/3复合物在封闭区的结构、调控和功能作用，有可能发现基因治疗或常规药物抑制骨吸收性疾病的新靶点。

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.