Actin Nucleation by Integrin Complexes

描述（由申请人提供）：此申请旨在为首席研究员提供额外的培训和保护时间，以便与当前NIAID资助的研究平行开发新的研究领域。我们对造血整合素受体激活的研究进展迅速，并与最近的其他报告一起表明，活化的β -3整合素复合体协调了肌动蛋白在应激纤维中的组装。这种新颖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数字显微镜的应用。正式的高级培训，在荧光显微镜和显著研究者时间将需要完成这些实验。活细胞成像和荧光显微镜的增加将是必要的，以适当地继续研究人员对造血细胞整合素介导的粘附和运动性的长期研究。

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.