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

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）椭球状成熟骨细胞。大量证据表明，骨细胞是直接调节成骨细胞和去骨细胞活性的关键机械传感细胞。因此，骨细胞的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，利用动态流体下骨细胞的生物传感器，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机械信号激活。提出的技术还结合了先进的计算流体动力学(CFD)-流体流动下的单元实体建模，用于估计“实时”单元粘弹性特性。考虑到流体流动、细胞变形和机械诱导Src/FAK激活的时间尺度（<0.3秒）的动态性，任何成像技术都必须具有能够在这些时间尺度上捕捉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，以更好地捕捉肌动蛋白和微管网络变形的空间动力学，Src或FAK激活，以及微图案，椭圆形骨细胞在振荡流下的全细胞力学特性。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、(2)利用振荡流体流动下骨细胞的伪三维成像，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变形和Src激酶或FAK激活，并关联局部定位生物传感器激活与网络应变。本研究将对骨生物学、细胞力学转导、细胞粘附以及多种贴壁细胞类型的力学生物学等领域的基础科学研究产生重大影响。

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.