Nanomechanics of intercellular adhesion

这项研究的主要目标是利用工程方法来确定控制组织中细胞间连接的组装和稳定性的基本机制和生物设计规则。这项研究的重点是一类蛋白质，钙粘蛋白，它是组织形成和维持所必需的。工程方法，包括纳米力学、细胞操作和表面化学修饰，将解决钙粘蛋白功能的三个关键方面，这是钙粘蛋白在发育生物学、组织形成和生物工程中的核心作用：具体来说，(1)选择性细胞间粘附的物理化学基础，(2)一类新的激活（和潜在治疗）抗体激活钙粘蛋白的分子机制和亲和力调节，以及(3)细胞间的机械转导以及钙粘蛋白亚型、粘附生物物理学和钙粘蛋白依赖的细胞内信号传导之间的联系。这项研究的智力价值在于由工程、生物物理学和细胞生物学方法的多学科结合所产生的新发现。这个PI的初步研究已经确定了钙粘蛋白介导的细胞间粘附的独特热力学和动力学特征。这一有利的发现将进一步确定不同的机制特征，这些特征被认为是细胞之间选择性粘附、亲和调节和这些基本粘附蛋白信号转导的基础。目的1利用这些发现i)确定钙粘蛋白粘附机制ii)量化影响选择性细胞间粘附的动力学和热力学参数。这些研究的变革力量在于它们能够识别在组织形成过程中指导细胞分离的钙粘蛋白键的物理化学性质（目的1）。相关研究将反过来确定最近分离的钙粘蛋白激活抗体如何增加（或调节）钙粘蛋白结合亲和力（目的2）。激活抗体具有巨大的治疗潜力，这些研究将定义钙粘蛋白粘附的生化调节剂的一般分子设计规则。最后，Aim 3扩展了PI的初步发现，即不同的钙粘蛋白引起不同的细胞内信号反应，这些信号反应似乎与蛋白质的结合强度无关。这一全新的结果构成了一个重大的范式转变，因为在四十年的研究中，试图确定钙粘蛋白特异性细胞相互作用的基础，只集中在细胞外结构域。Aim 3利用这一独特的发现，将在确定调节组织（钙粘蛋白）特异性细胞功能的关键生物设计规则方面开辟新天地。该方案的广泛影响源于其与生物工程和人类健康的直接关系，以及通过在大学预科和本科阶段向妇女和少数民族提供服务而产生的教育影响。钙粘蛋白是组织发生所必需的，它们维持所有实体组织的结构完整性；因此，该计划的结果将影响生物工程和基础细胞生物学的几个领域，包括神经发育，胚胎发生，癌症，肠上皮疾病，血管渗漏疾病，伤口愈合，组织再生，干细胞分化和组织工程。例如，在与弗吉尼亚大学医学院的B. Gumbiner的合作中，研究小组将独特地确定通过新型钙粘蛋白激活抗体调节钙粘蛋白亲和力的机制，这种抗体可能治疗广泛的钙粘蛋白相关疾病。钙粘蛋白结合和细胞内信号传导的研究将进一步确定在组织形成、修复和再生过程中可能有助于细胞分离、组织模式和可能的组织特异性功能的物理和生化机制。这些研究还将确定在组织支架和工程环境中模拟细胞-细胞相互作用所需的生化和生物物理参数。本提案中描述的一些技术也将被用作向芝加哥地区高中的中学女生、少数族裔本科生、女性和少数族裔开展推广活动的工具。在与伊利诺伊大学外展项目的合作中，PI试图改变初高中学生对科学和科学事业的先入为主的观念。她还将使用本提案中描述的一些研究活动来指导本科生参加科学和工程领域代表性不足的少数民族的暑期研究项目。该项目还将继续支持培训来自科学和工程领域代表性不足群体的学生。PI指导了16名毕业论文学生，其中约40%是女性。在研究生阶段，PI指导了2名女性硕士研究生，9名女性博士研究生，2名非洲裔美国人博士研究生和1名西班牙裔男性博士研究生。该项目提供及时的研究机会，将继续吸引不同的学生，并提供从大学预科到研究生教育水平的研究和工程方面的优质机会。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)0853705LeckbandThe broad goal of this research is to use engineering approaches to identify fundamental mechanisms and biological design rules that control the assembly and stability of intercellular junctions in tissues. This research focuses on a class of proteins, cadherins, which are essential for the formation and maintainenance of organized tissues. Engineering approaches, including nanomechanics, cell manipulation, and surface chemical modification will address three key aspects of cadherin function that are central to cadherin's role in developmental biology, tissue formation, and bioengineering: specifically, (1) the physical chemical basis of selective cell-to-cell adhesion, (2) molecular mechanism(s) of cadherin activation and affinity modulation by a novel class of activating (and potentially therapeutic) antibodies, and (3) mechanical transduction between cells and the link between cadherin subtype, adhesion biophysics, and cadherin-dependent intracellular signaling.The intellectual merit of this research lies in the novel discoveries engendered by this multidisciplinary combination of engineering, biophysics, and cell biological approaches. Preliminary studies by this PI already identified unique thermodynamic and kinetic signatures of cadherin-mediated cell-to-cell adhesion. This enabling discovery will further the identification of distinguishing mechanistic features that are postulated to underlie the selective adhesion between cells, affinity regulation, and signal transduction by these essential adhesion proteins. Aim 1 exploits these findings i) to identify mechanisms of cadherin adhesion and ii) to quantify kinetic and thermodynamic parameters that influence selective cell-to-cell adhesion. The transformative power of these investigations lies in their capacity to identify the physical chemical properties of cadherin bonds postulated to direct cell segregation during tissue formation (Aim 1). Related investigations will in turn identify how recently isolated, cadherin-activating antibodies increase (or modulate) cadherin binding affinity (Aim 2). Activating antibodies have tremendous therapeutic potential, and these studies will define general, molecular design rules for biochemical modulators of cadherin adhesion. Finally, Aim 3 extends the PI's preliminary findings that different cadherins elicit distinct intracellular signaling responses that are seemingly independent of the protein bond strengths. This entirely novel result constitutes a major paradigm shift, because in four decades of research, attempts to determine the basis of cadherin-specific cell interactions exclusively focussed on the extracellular domains. Aim 3 exploits this unique discovery, and will break new ground in identifying key biological design rules that regulate tissue (cadherin)-specific cell functions. The broad impact of this program derives from its direct relevance to bioengineering and human health and from its educational impact through outreach to women and minorities at both the pre-college and undergraduate levels. Cadherins are essential for tissue genesis and they maintain the structural integrity of all solid tissues; consequently, the results of this program will impact several areas of bioengineering and basic cell biology, including, neural development, embryogenesis, cancer, diseases of the intestinal epithelium, vascular leakage diseases, wound healing, tissue regeneration, stem cell differentiation, and tissue engineering. For example, in collaboration with B. Gumbiner at the University of Virginia Medical School, the research team will uniquely determine the mechanism(s) of cadherin affinity modulation by novel, cadherin-activating antibodies, which could potentially treat a wide range of cadherin-related diseases. Investigations of cadherin binding and intracellular signaling will further identify physical and bio-chemical mechanisms that may contribute to cell segregation, tissue patterning, and possibly tissue-specific functions during tissue formation, repair, and regeneration. These investigations will also identify biochemical and biophysical parameters that are required to mimic cell-cell interactions in tissue scaffolds and engineered environments.Several of the technologies described in this proposal will also be used as vehicles for outreach activities to middle school girls, minority undergraduates, and women and minorities in Chicago area high schools. In partnerships with outreach programs at the University of Illinois, the PI seeks to alter preconceptions of science and scientific careers often held by middle- and high-school students. She will also use some of the research activities described in this proposal to mentor undergraduates participating in summer research projects for underrepresented minorities in science and engineering.This program will also continue to support the training of students from underrepresented groups in science and engineering. The PI has mentored 16 senior thesis students, approximately 40% of which were women. At the graduate level, the PI mentored 2 female Masters candidates, 9 female PhD candidates, 2 African American PhD candidates, and one Hispanic male PhD student. This program offers timely research opportunities that will continue to attract diverse students and provide quality opportunities in research and engineering from pre-college to graduate educational levels.