Full Sail Ahead: How Membranes Move and Respond to Flow

项目总结 脂膜的一个显著特征是它们的流动性：它们可以自我修复、弯曲和循环。 个别细胞也体验并响应其环境中的流动。流动反应调节不同的 血压、骨密度和神经生长等过程。这一点在血液中尤为明显 血管，其中单层内皮细胞形成流动的血液和静止的血液之间的界面 组织。低血流区域和动脉粥样硬化斑块之间的相关性在一个世纪前就被观察到了， 从而得出剪切流影响内皮细胞功能的假说。了解细胞如何完成任务 剪切应力向细胞信号的机械传递引起了人们的广泛关注。然而，分子 流动机械转导背后的决定因素仍不清楚。特别是，我们缺乏关于侧向的信息 位于细胞-流体界面的胞外膜蛋白的运动。在观察到流动的同时 对于膜蛋白的转运，这种转运如何影响蛋白质的功能和细胞反应仍是个未知数。 未知。 拟议研究的目标是定量测量特定于脂质的物理相互作用。 决定脂质和蛋白质如何响应流动的膜，并测试流动是否 膜蛋白激活内皮细胞的细胞内信号。我们的中心假设是 生理上显著的蛋白质和脂肪浓度梯度产生于 流体流动和复合膜。这一假说是基于这样一个前提，即细胞外脂锚定 像Glypcan-1这样的蛋白多糖可以通过外流沿着质膜运输，与 蛋白质的水部分，起分子帆的作用。我们将实现三个具体目标：一是 确定控制膜连接蛋白流动运输的基本性质和原理。 为膜建立模型，并建立一个模型来预测生理环境中的蛋白质运动。在第二个目标中， 我们将确定一种生理上重要的膜蛋白如何通过流动介导的侧向运输 (GLYPICAN-1)启动内皮细胞的短期流动反应。在我们的第三个目标中，我们将调查 在我们的模型系统和活细胞膜中，按流进行的脂质分类有助于流信号的传递。 我们的方法是在模型膜和活细胞中进行平行实验，允许我们 将生理功能与分子生物物理学直接联系起来。实验依赖于PI的专业知识，使用 实验微流体学和共聚焦显微镜，以确定膜的基本性质。而当 这里研究的模型蛋白是针对内皮细胞的，我们将揭示流体力学的原理 是普遍存在的。因此，我们预计我们的模型将适用于多个细胞系和流动条件，并且 将为今后的研究方向奠定基础。

PROJECT SUMMARY A remarkable feature of lipid membranes is their fluidity: they can self-heal, bend, and circulate. Individual cells also experience and respond to the flows in their environment. Flow responses regulate diverse processes such as blood pressure, bone density, and neural growth. This is particularly apparent in blood vessels, where a monolayer of endothelial cells forms the interface between flowing blood and stationary tissue. Correlation between regions of low flow and atherosclerotic plaques was observed a century ago, leading to the hypothesis that shear flow impacts endothelial cell function. Understanding how cells accomplish mechanotransduction of shear stress into cellular signals is of wide interest. However, the molecular determinants behind flow mechanotransduction remain unclear. Particularly, we lack information on the lateral movement of extracellular membrane proteins located at the cell-fluid interface. While flow has been observed to transport membrane proteins, how this transport affects protein function and cell responses remains unknown. The goal of the proposed studies is to quantitatively measure the physical interactions specific to lipid membranes that determine how lipids and proteins move in response to flow and test whether flow transport of a membrane protein activates intracellular signaling in endothelial cells. Our central hypothesis is that physiologically significant protein and lipid concentration gradients arise from physical interactions between fluid flow and complex membranes. This hypothesis is based on the premise that extracellular lipid-anchored proteoglycans like glypican-1 can be transported along the plasma membrane by external flow, with the aqueous part of the protein acting as a molecular sail. We will accomplish three specific aims: Our first aim is to identify the fundamental properties and principles that govern flow transport of membrane-linked proteins in model membranes and to build a model to predict protein motion in physiological contexts. In the second aim, we will determine how the flow-mediated lateral transport of a physiologically important membrane protein (glypican-1) initiates the short-term flow response in endothelial cells. In our third aim, we will investigate how lipid sorting by flow contributes to flow signaling in our model system and living cell membranes. Our approach is to conduct parallel experiments in model membranes and living cells, allowing us to directly relate physiological function to molecular biophysics. The experiments rely on the PI's expertise using experimental microfluidics and confocal microscopy to determine fundamental membrane properties. While the model protein studied here is specific to endothelial cells, the principles of fluid mechanics that we will uncover are universal. We, therefore, anticipate that our models will apply to multiple cell lines and flow conditions, and will lay the groundwork for future research directions.