Pseudo-3D Cytoskeleton Dynamics and Signal Activation in Osteocytes under Flow

流动下骨细胞的伪 3D 细胞骨架动力学和信号激活

• 批准号: 7978377
• 负责人: X. Edward GUO
• 金额: $ 18.17万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7978377
• 关键词: 3-Dimensional; Actins; Address; Adhesions; Apical; Area; Basic Science; Biochemical; Biology; Biosensor; Bone Tissue; Cell Adhesion; Cell model; Cell physiology; Cells; Cellular Mechanotransduction; Cellular biology; Complex; Computer Simulation; Cytoskeleton; Dendrites; Etiology; Event; Extracellular Matrix; Fluorescence Resonance Energy Transfer; Focal Adhesion Kinase 1; Frequencies; Grant; Health; Image; Imaging Techniques; Individual; Liquid substance; Mature Bone; Measures; Mechanical Stimulation; Mechanics; Microfilaments; Microscopy; Microtubules; Modeling; Morphology; Nature; Osteoblasts; Osteoclasts; Osteocytes; Osteoporosis; Pathway interactions; Phosphotransferases; Physiological; Play; Printing; Process; Property; Research; Resolution; Role; Scanning; Shapes; Signal Transduction; Solid; Stimulus; Stress; Surface; Techniques; Technology; Testing; Time; base; bone; bone cell; cell motility; cell type; fluid flow; high risk; in vivo; migration; monolayer; neuronal cell body; novel; public health relevance; response; shear stress; src-Family Kinases; time use

DESCRIPTION (provided by applicant): Osteocytes are three-dimensional (3D) ellipsoidal shaped mature bone cells encased in mineralized extracellular matrix. Abundant evidence has shown that the osteocytes are the key mechanonsensor cells that directly regulate bone-forming osteoblast and bone-removing osteoclast activities. Thus, osteocyte functions are critical to the etiology and treatments of osteoporosis, one of the most challenging health issues in the U.S. The proposed project will develop a pseudo-3 dimensional (3D) real-time microscopy technology to simultaneously visualize cell deformation, actin and microtubule cytoskeleton dynamics, and mechano- signaling activation in real-time using biosensors of osteocyte cells under dynamic fluid flow. The proposed technology also incorporates advanced computational fluid dynamics (CFD)-solid modeling of a cell under fluid flow for the estimation of "real-time" cell viscoelastic properties. Given the dynamic nature of fluid flow, cell deformation, and the timescale of mechanically-induced Src/FAK activation (<0.3 seconds), any imaging technique must have a temporal resolution capable of capturing cell deformation at these timescales. Here, we propose using a real-time "pseudo-3D" technique to image two orthogonal views of a cell simultaneously to greater capture the spatial dynamics of actin and microtubule network deformation, Src or FAK activation, and whole cell mechanical properties of micropatterned, ellipsoidally shaped osteocytes under oscillatory flow. The specific aims of the project are: (1) Simultaneously track and analyze individual cell deformation, actin filament networks, and microtubule networks using pseudo-3D imaging of osteocytes under oscillatory fluid flow and correlate the predicted whole cell mechanical properties with the intracellular actin filament or microtubule network strains and (2) Simultaneously track intracellular actin or microtubule network deformation and Src kinase or FAK activation by fluorescence resonance energy transfer (FRET) biosensors using pseudo-3D imaging of osteocytes under oscillatory fluid flow and correlate the localized biosensor activation with the network strains. This study will have significant impact in the basic science research in the field of bone biology, cellular mechanotransduction, cell adhesion, and the mechanobiology of many adherent cell types. PUBLIC HEALTH RELEVANCE: This R21 application will develop a real-time "pseudo-3D" microscopy technique to image two orthogonal views of a cell under dynamic fluid flow simultaneously to greater capture the spatial dynamics of cytoskeletal deformation, Src or FAK activation, and whole cell mechanical properties of osteocytes. This enabling technology will have great impact in the general fields of bone biology and cellular mechanics for an increased understanding of the role that physical forces play in bone cell mechanotransduction.

描述（由申请人提供）：骨细胞是包裹在矿化细胞外基质中的三维（3D）椭圆形成熟骨细胞。大量证据表明，骨细胞是直接调节成骨细胞和破骨细胞活动的关键力学感受器细胞。因此，骨细胞功能对于骨质疏松症的病因学和治疗至关重要，骨质疏松症是美国最具挑战性的健康问题之一。拟议的项目将开发一种伪三维（3D）实时显微镜技术，以同时可视化细胞变形，肌动蛋白和微管细胞骨架动力学，以及使用动态流体流动下骨细胞生物传感器实时机械信号激活。所提出的技术还结合了先进的计算流体动力学（CFD）-流体流动下的细胞的实体建模，用于估计“实时”细胞粘弹性。考虑到流体流动、细胞变形和机械诱导的Src/FAK激活的时间尺度（<0.3秒）的动态性质，任何成像技术必须具有能够在这些时间尺度下捕获细胞变形的时间分辨率。在这里，我们建议使用一个实时的“伪3D”技术，图像的两个正交视图的细胞，同时更大的捕获肌动蛋白和微管网络变形，Src或FAK激活，和整个细胞的力学性能的微图案，椭圆形的骨细胞振荡流下的空间动态。该项目的具体目标是：（1）同时跟踪和分析单个细胞变形，肌动蛋白丝网络，和微管网络，并将预测的全细胞机械特性与细胞内肌动蛋白丝或微管网络应变相关联，以及（2）同时跟踪细胞内肌动蛋白或微管网络变形和Src激酶或FAK激活的荧光共振能量转移（FRET）生物传感器使用伪-振荡流体流动下骨细胞的3D成像，并将局部生物传感器激活与网络应变相关联。本研究将对骨生物学、细胞力学传导、细胞粘附以及多种粘附细胞类型的力学生物学等领域的基础科学研究产生重要影响。 公共卫生关系：该R21应用程序将开发一种实时“伪3D”显微镜技术，以同时在动态流体流动下对细胞的两个正交视图进行成像，以更好地捕获细胞骨架变形、Src或FAK激活以及骨细胞的全细胞机械特性的空间动态。这种使能技术将在骨生物学和细胞力学的一般领域产生巨大的影响，以增加对物理力在骨细胞机械转导中所起作用的理解。