Using Nanotechnology to Regulate Integrin Clustering and Tensin Localization

利用纳米技术调节整合素聚类和张力蛋白定位

• 批准号: 7114754
• 负责人: GANG-YU LIU
• 金额: $ 18.69万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-29 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7114754
• 关键词: actin binding protein; atomic force microscopy; binding sites; biological signal transduction; biotechnology; cell adhesion; cell line; cell migration; cell motility; confocal scanning microscopy; fibronectins; immunofluorescence technique; integrins; method development; molecular /cellular imaging; molecular assembly /self assembly; nanotechnology; protein localization; protein structure; scanning electron microscopy

DESCRIPTION (provided by applicant): The long-term goal is to develop nanobiotechnology that allows the understanding of the initial signal transduction process in time and in space, and with that knowledge to devise strategies to alter the behavior of cells in general, with focus on cancer cells in specific. The idea is based on the observation that clustering of integrin and other focal proteins such as tensin are correlated to the downstream cell signaling. By varying the geometric parameters of nanostructures of integrin-binding sites, such as size, shape, and separation, the localization of tensin at focal adhesion would change accordingly, thus altering the corresponding cell signaling pathways. The two co-PIs have complementary expertise in nanotechnology (Liu) and cell biology of focal adhesion involving tensin (Lo), respectively. Preliminary studies from the co-PI (Lo) laboratory reveal that tensin localization at focal adhesion impacts on corresponding cell functions such as motility and migration. Preliminary study from the PI (Liu) group has demonstrated that scanning probe microscopy based nanofabrication enables precise positioning of simple molecular ligands, proteins and DNA on surfaces. The first goal is to develop a new methodology, combined atomic force and confocal microscopy for single cell imaging and for visualization of protein complexes at focal adhesion. Confocal microscopy will be utilized to (a) reproduce previously known immunofluorescence studies in order to validate this new method; and (b) to guide atomic force microscopy (AFM) probes to penetrate into cells to reach focal adhesion sites for high resolution imaging of protein complexes. Arrays of nanostructures consisting of integrin-binding ligands such as fibronectin will be produced with designed size and geometry using Liu's nanofabrication methodologies. Finally, NIH 3T3 cells will be seeded onto these nanostructure arrays where integrin/tensin clusters will form, and be characterized using this combined AFM/confocal microscopy to reveal how the tensin localization varies as a function of the ligand nanostructures underneath. Once this concept of regulating tensin translocation via nanotechnology is proven, future work can be planned to investigate how cell functions can be regulated by forming designed integrin clusters and tensin location using ligand nanostructures underneath the cells. Formation of other protein complexes such as tyrosine kinases at focal adhesion may be regulated following the same concept. This nanoengineering approach should provide a new paradigm for controlling cell signaling and behavior, which enables new methods be developed for cancer therapy.

描述（由申请人提供）：长期目标是开发纳米型技术，以了解时间和空间中的初始信号转导过程，并以这些知识来制定策略以改变细胞的行为，并侧重于特定的癌细胞。该想法基于这样的观察结果，即整联蛋白和其他焦点蛋白（如tensin）的聚类与下游细胞信号传导相关。通过改变整联蛋白结合位点的纳米结构的几何参数（例如大小，形状和分离），在焦点粘附处的Tensin的定位将相应地改变，从而改变相应的细胞信号传导途径。这两个Co-Pis分别具有纳米技术（LIU）和细胞生物学的互补专业知识，分别涉及Tensin（LO）（LO）。 CO-PI（LO）实验室的初步研究表明，局灶性粘附处的紧张定位会影响相应的细胞功能，例如运动和迁移。 PI（LIU）组的初步研究表明，扫描基于探针显微镜的纳米化表现可以使简单分子配体，蛋白质和DNA在表面上精确定位。第一个目标是开发一种新的方法论，将原子力和共聚焦显微镜组合起来，用于单细胞成像以及在局灶性粘附时可视化蛋白质复合物。共聚焦显微镜将用于（a）再现先前已知的免疫荧光研究，以验证这种新方法； （b）引导原子力显微镜（AFM）探针渗透到细胞中以达到局灶性粘附位点，以进行蛋白质复合物的高分辨率成像。使用LIU的纳米纳米制作方法，将生产由整联蛋白结合配体（例如纤连蛋白）组成的纳米结构阵列。最后，将NIH 3T3细胞接种到这些纳米结构阵列中，在这些纳米结构阵列中，整联蛋白/Tensin簇将形成，并使用此组合的AFM/共聚焦显微镜进行表征，以揭示Tensin定位如何随着下面的配体纳米结构的功能而变化。一旦证明了通过纳米技术调节Tensin易位的概念，就可以计划使用未来的工作来研究细胞功能如何通过使用细胞下方的配体纳米结构形成设计的整合素簇和Tensin位置来调节细胞功能。可以按照相同的概念来调节其他蛋白质复合物，例如局灶性粘附处的酪氨酸激酶。这种纳米工程方法应为控制细胞信号传导和行为提供新的范式，从而为癌症治疗开发新方法。