Actin Nucleation by Integrin Complexes

整合素复合物使肌动蛋白成核

• 批准号: 7373569
• 负责人: SCOTT D BLYSTONE
• 金额: $ 11.37万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373569
• 关键词: Actins; Adhesions; Affect; Area; Biochemical; Biochemistry; Biological Assay; Cell Adhesion; Cells; Cellular Structures; Color; Complex; Condition; Electron Microscopy; Event; Exhibits; F-Actin; Failure; Fiber; Filopodia; Funding; Hematopoietic; Imagery; In Vitro; Integrin beta3; Integrin-mediated Cell Adhesion Pathway; Integrins; Investigation; Leukocytes; Life; Ligands; Localized; Longitudinal Studies; Membrane; Methods; Microscopy; Modeling; Molecular; Mutation; National Institute of Allergy and Infectious Disease; Participant; Personal Satisfaction; Phase; Physiological; Principal Investigator; Production; Proteins; Receptor Activation; Regulation; Reporting; Research; Research Personnel; Signal Transduction; Site; Standards of Weights and Measures; Stress Fibers; Structure; Supporting Cell; System; Technology; Time; Training; Tyrosine Phosphorylation; cell motility; cellular imaging; digital; in vitro Assay; mutant; novel; polymerization; research study; size; theories

DESCRIPTION (provided by applicant): This application is intended to provide additional training and protected time for the principal investigator to develop a new area of research in parallel to current NIAID funded research. Our efforts to describe the activation of hematopoietic integrin receptor activation has progressed rapidly and, along with other recent reports, now suggests that activated beta-3 integrin complexes orchestrate the assembly of actin into stress fibers. This novel hypothesis requires the use of a recently developed in vitro assay of actin assembly and the application of advanced two-color live digital microscopy. Formal advanced training in fluorescent microscopy and significant investigator time will be required for the completion of these experiments. The addition of live cell imaging and fluorescent microscopy will be necessary to appropriately continue the investigators long-term study of hematopoietic cell integrin-mediated adhesion and motility. Current theory of cell motility and adhesion proposes localized actin assembly leading to membrane deformation. This would be followed by integrin interaction first with matrix ligand and then with cytosolic F-actin. Recently, actin nucleating proteins were shown to associate with integrin complexes. We have developed an in vitro assay for actin assembly by purified hematopoietic integrins. This assay reflects deficiencies in cell adhesion through quantitative and qualitative assessment of actin polymerization and was developed to investigate the failure of a Tyr747Phe mutant ?3 integrin to support cell adhesion. Current NIAID funding supports continuing investigation into the signaling mechanisms and physiologic relevance of ?3 tyrosine phosphorylation, but does not provide support for determining integrin control of cytoskeletal assembly. We hypothesize that integrin activation sequesters actin nucleating proteins and that the activated integrin complex serves as the origin of actin fiber assembly during stress fiber formation. If true, this will join integrin signaling and actin assembly fields in a unique approach to understanding cell adhesion and motility. Utilizing integrins purified from hemaotpoietic cells, that exhibit an inactive basal state and applying technology from the actin biochemistry field we will establish in vitro actin stress fiber assembly as an integrin function and use it to understand integrin regulation of cell adhesion and motility.

描述（由申请人提供）：本申请旨在为主要研究者提供额外的培训和保护时间，以开发与当前NIAID资助的研究平行的新研究领域。我们对造血整合素受体激活的研究进展迅速，沿着其他近期报道，现在表明激活的β-3整合素复合物协调肌动蛋白组装成应力纤维。这一新的假设需要使用最近开发的肌动蛋白组装的体外测定和先进的双色活体数字显微镜的应用。完成这些实验需要荧光显微镜的正式高级培训和大量的研究时间。增加活细胞成像和荧光显微镜将是必要的，以适当地继续研究人员的造血细胞整合素介导的粘附和运动的长期研究。 目前的细胞运动和粘附理论提出了局部肌动蛋白组装导致膜变形。这将其次是整合素相互作用，首先与基质配体，然后与胞质F-肌动蛋白。最近，肌动蛋白成核蛋白被证明与整合素复合物。我们已经开发了一种体外测定肌动蛋白组装纯化造血整合素。该检测反映了细胞粘附的缺陷，通过定量和定性评估肌动蛋白聚合和研究失败的Tyr 747 Phe突变？3整合素以支持细胞粘附。目前NIAID的资金支持继续调查的信号机制和生理相关性？3酪氨酸磷酸化，但不提供支持，确定整合素控制的细胞骨架组装。我们推测，整合素激活螯合肌动蛋白成核蛋白，激活的整合素复合物作为应力纤维形成过程中肌动蛋白纤维组装的起源。如果是真的，这将加入整合素信号和肌动蛋白组装领域的一个独特的方法来理解细胞粘附和运动。 利用从造血细胞中纯化的整合素，表现出无活性的基础状态，并应用肌动蛋白生物化学领域的技术，我们将建立体外肌动蛋白应力纤维组装作为整合素功能，并使用它来了解整合素对细胞粘附和运动的调节。