Systems mechanisms underlying cell shape based phenotypic switches

 描述（由申请人提供）：在本提案中，我们希望了解3D细胞形状在血管平滑肌细胞（VSMC）表型状态中的作用。我们将使用不同3D形状的血管平滑肌细胞网络的数值建模和微图案中的高分辨率显微镜进行实验验证。研究细胞形状对VSMC表型的作用将影响我们对动脉粥样硬化等血管疾病起源的理解，以及细胞形状如何调节许多细胞类型的表型状态。将VSMC微图案化成3D形状提供了一个独特的机会，将细胞形状的影响与其他因素（如细胞与细胞的接触和细胞外基质）分离开来，这些因素可能会合并细胞形状的影响。为了确定细胞形状改变VSMC中钙信号传导和基因表达的机制，我们有三个重叠的目标。1)进行VSMC的数值模拟。我们将使用虚拟细胞或COMSOL开发受体-配体相互作用及其下游效应物的多房室ODE模型。我们将研究毒蕈碱受体通路（M3 R）、TGF和PDGF信号传导及其生理反应，包括细胞形状对Ca 2+和转录因子活性的不同影响。VSMC将被接种到3D微加工表面，在那里它们可以模拟主动脉组织中层中VSMC的组织。我将使用活细胞成像，荧光相关光谱和超分辨率显微镜来表征受体，钙信号和转录因子活动在椭圆形和球形的质膜分布。3)在3D微加工表面上对VSMC进行mRNAseq实验。为了对细胞形状引发的变化进行公正的观察，我们将通过mRNAseq分析基因表达，并使用差异表达基因产物的免疫荧光来确认结果。我们将确定在生理学中发挥选择性作用的基因，并通过siRNA消融实验测试其效果。

 DESCRIPTION (provided by applicant): In this proposal we would like to understand the role of 3D cell shape in the phenotypic status of vascular smooth muscle cells (VSMC). We will be using a combination of numerical modeling of vascular smooth muscle cell network in different 3D shapes and high resolution microscopy in micropatterns for experimental validation. Studying the role of cell shape on the phenotypes of VSMC will impact our understanding of the origins of vascular diseases such as atherosclerosis and how cell shape might regulate phenotypic states in many cell types. Micropatterning VSMC into 3D shapes provides a unique opportunity to segregate the effect of cell shape from other factors such as cell to cell contacts and extracellular matrix that could conflate cell shape effects. To identify the mechanisms by which cell shape changes calcium signaling and gene expression in VSMC, we have three overlapping aims. 1) To conduct numerical simulations of VSMC . We will develop a multi-compartmental ODE model of receptor-ligand interactions and their downstream effectors using Virtual Cell or COMSOL. We will study muscarinic receptor pathway (M3R), TGF and PDGF signaling and their physiological responses including Ca2+ and transcription factor activities which are differentially impacted by cell shape based on our preliminary data 2) To conduct experiments on VSMC in 3D microfabricated surfaces. VSMC will be seeded to 3D microfabricated surfaces where they could mimic the organization of VSMC in a medial layer of aortic tissue. I will characterize the plasma membrane distribution of receptors, calcium signaling and transcription factor activities in ellipsoid and spherical shapes using live cell imaging, fluorescence correlation spectroscopy and super-resolution microscopy. 3) To Conduct mRNAseq experiments on VSMC in 3D microfabricated surfaces. For an unbiased view of the changes triggered by cell shape, we will profile gene expression by mRNAseq and confirm results using immunofluorescence of differentially expressed gene products. We will identify genes that play a selective role in physiology and test their effects by siRNA ablation experiments.