Actin Nucleation by Integrin Complexes

描述(由申请人提供)：本申请旨在为首席研究员提供额外的培训和受保护的时间，以便在NIAID资助的当前研究的同时开发一个新的研究领域。我们对造血整合素受体激活的研究进展迅速，与最近的其他报道一起，现在表明激活的β-3整合素复合体协调肌动蛋白组装成应激纤维。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试和先进的双色活数字显微镜的应用。完成这些实验需要正规的高级荧光显微镜培训和大量的研究人员时间。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对造血细胞整合素介导的黏附和运动的长期研究。 目前的细胞运动和黏附理论认为，肌动蛋白的局部组装导致了膜的变形。紧随其后的是整合素首先与基质配体相互作用，然后与胞浆F-肌动蛋白相互作用。最近，肌动蛋白核蛋白被证明与整合素复合体相关。我们建立了一种利用纯化的造血整合素进行肌动蛋白组装的体外检测方法。通过对肌动蛋白聚合的定量和定性评估，该方法反映了细胞黏附的缺陷，并被开发用于研究Tyr747Phe突变体？3整合素未能支持细胞黏附。目前的NIAID资金支持继续研究β3酪氨酸磷酸化的信号机制和生理相关性，但不支持确定整合素对细胞骨架组装的控制。我们假设整合素激活隔离了肌动蛋白核蛋白，并且激活的整合素复合体在应力纤维形成过程中是肌动蛋白纤维组装的起源。如果这是真的，这将加入整合素信号和肌动蛋白组装领域，以一种独特的方法来了解细胞黏附和运动性。 利用从基础状态不活跃的造血细胞中提纯的整合素，并应用肌动蛋白生物化学领域的技术，我们将在体外建立作为整合素功能的肌动蛋白应激纤维组装，并利用它来了解整合素对细胞黏附和运动的调节。

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.