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

描述(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去骨破骨细胞活动的关键机械敏感细胞。因此，骨细胞功能对骨质疏松症的病因和治疗至关重要，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，以同时显示细胞变形、肌动蛋白和微管细胞骨架动力学以及在动态流体流动下使用骨细胞生物传感器实时激活的机械信号。该技术还结合了先进的计算流体力学(CFD)--流体流动下细胞的固体建模，用于估计细胞的“实时”粘弹性性质。考虑到流体流动、细胞变形的动态性质，以及机械诱导的Src/FAK激活的时间尺度(&lt；0.3秒)，任何成像技术都必须具有能够捕捉这些时间尺度上的细胞变形的时间分辨率。在这里，我们建议使用实时“伪3D”技术同时对细胞的两个正交视图进行成像，以更好地捕捉振荡流动下微图案化、椭球体形状的骨细胞的肌动蛋白和微管网络变形、Src或FAK激活以及整个细胞力学特性的空间动力学。该项目的具体目标是：(1)利用振荡流体下骨细胞的伪3D成像同时跟踪和分析单个细胞的变形、肌动蛋白细丝网络和微管网络，并将预测的整个细胞的力学特性与细胞内肌动蛋白细丝或微管网络应变相关联；(2)利用振荡流体下骨细胞的伪3D成像技术，利用荧光共振能量转移(FRET)生物传感器同时跟踪细胞内肌动蛋白或微管网络的变形和Src激酶或FAK的激活，并将生物传感器的局部激活与网络应变相关联。该研究将对骨生物学、细胞力学转导、细胞黏附以及多种黏附细胞的力学生物学等基础科学研究产生重大影响。 与公共健康相关：这个R21应用程序将开发一种实时“伪3D”显微镜技术，以同时对动态流体流动下细胞的两个正交视图进行成像，以更好地捕捉细胞骨架变形、Src或FAK激活以及骨细胞的全细胞力学特性的空间动力学。这项使能技术将对骨生物学和细胞力学的一般领域产生重大影响，以加深对物理力在骨细胞机械转导中所起作用的了解。

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.