Imaging and Quantifying Lipid Membrane Asymmetry in Living Cells with Sum-Frequency Vibrational Microscopy

在这个由化学系化学结构、动力学和机制A(CSDM-A)、生命过程化学(CLP)和化学测量和成像(CMI)计划资助的项目中，犹他大学的John Conball教授和Markus Babst教授正在探索细胞膜的结构。细胞膜由各种类型的分子组成，但它们的主要成分是脂分子，脂分子本身由避免与水接触的长油状碳氢链组成(疏水)，一端含有磷和或氧原子，使这一端倾向于在水存在下(亲水)。细胞膜实际上是两层脂分子；两层上的长疏水链在膜内相互面对，亲水的头部基团暴露在细胞内外的水环境中。在活细胞中，膜可以是不对称的；换句话说，内层和外层的脂类的数量和具体种类是不同的。目前的细胞膜结构理论认为，这种不对称性是由于存在于细胞膜上的另一类分子的作用，这些分子被称为Floppase和Floppase。这些蛋白质被认为控制着脂质分子从细胞膜的一边到另一边的运动。康博伊教授和巴布斯特教授假设，如果膜中没有翻转酶和折叠酶，只需通过与细胞内蛋白质的内壁相互作用，就可以出现膜不对称。为了验证他们的假设，康博伊教授和巴布斯特教授正在使用一种名为和频振动显微镜的特殊技术，这种技术可以揭示膜中脂分子的分子结构，以及这些分子从一层到另一层的运动。该项目的目标之一是首次直接测量并生成活细胞中脂质不对称的图像。这项研究项目试图揭示大的复杂分子如何相互作用形成更大的组装结构的新的基本见解。这个项目的发现可能会影响我们对生命系统的看法。该项目是对两名研究生和两名本科生进行高级培训的工具。除了正式的培训活动外，康博伊和巴布斯特研究小组还参与公共推广活动，将科学带给更广泛的受众。我们目前对细胞膜的看法是由辛格和尼科尔森在20世纪70年代初由S建立的。他们提出的流体马赛克模型将膜描绘成一个由脂类、胆固醇和蛋白质组成的“液体状”双层，在平行于膜表面的两个维度上显示出快速和自由的扩散。与之形成鲜明对比的是，双层的小叶之间的脂类交换被推测为被与通过疏水膜核心的亲水性脂头基团转移相关的大的能量障碍所阻止。这种静态的脂质移位(或触发器)图像一直是膜动力学研究中的一个长期信念，也是目前关于细胞膜成分，特别是磷脂酰丝氨酸(PS)不对称的双边组织理论的基础。我们假设自发的PS触发器在活体中是一个常见的、简单的过程。这一预测提出了一个问题：细胞如何在质膜上维持PS的不对称性？我们的假设是，在体内PS定位到细胞膜的胞质小叶是由与细胞质蛋白质，特别是组成细胞骨架的蛋白质的静电相互作用驱动的。我们提出的PS不对称性模型与目前公认的观点不同之处在于，我们的模型提出了一个非常动态的膜系统，而不是更传统的“静态”脂质不对称性。这种动态行为改变了目前关于脂类不对称的产生和维持的理论；然而，它也简化和统一了对许多已知脂类现象的解释。我们的假设正在通过构建一种独特的和频振动显微镜来验证，该显微镜能够直接定量和成像活细胞中的脂质不对称，并首次在体内测量天然的脂类触发器。参与这个项目的学生正在获得严格的物理和生物物理概念以及基于非线性光学过程的先进成像技术的知识和经验。外展活动包括在盐湖谷参与K-12学校，特别是为比例较高的代表不足的群体提供服务的天主教学校。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

In this project funded by the Chemical Structure, Dynamics and Mechanisms A (CSDM-A), Chemistry of Life Processes (CLP) and Chemical Measurement and Imaging (CMI) Programs of the Chemistry Division, Professors John Conboy and Markus Babst of the University of Utah are exploring the structure of cell membranes. Cell membranes are composed of various types of molecules, but their major constituents are lipid molecules, which themselves are composed of long oily hydrocarbon chains that avoid contact with water (hydrophobic), and one end that contains phosphorus and or oxygen atoms that make that end prefer to be in the presence of water (hydrophilic). Cell membranes are actually two layers of lipid molecules; the long hydrophobic chains on the two layers face each other inside the membrane, and the hydrophilic head groups are exposed to watery environments inside and outside the cell. In living cells, the membrane can be asymmetric; in other words, the number and specific kinds of lipids in the inner and outer lipid layers are different. The current theory of cell membrane structure assumes that this asymmetry is due to the action of another class of molecules present in cell membranes called flippases and floppases. These are proteins that are thought to control movement of lipid molecules from one side of the cell membrane to the other. Professors Conboy and Babst hypothesize that membrane asymmetry can arise without flippases and floppases in the membrane, and simply through inner membrane wall interactions with proteins inside the cell. In order to test their hypothesis, Professors Conboy and Babst are using a special technique called sum-frequency vibrational microscopy that can reveal the molecular structure of the lipid molecules in a membrane as well as the motion of these molecules from one layer to the other. One goal of the project is to directly measure and generate images of lipid asymmetry in living cells for the first time. This research project seeks to reveal new fundamental insights into how large complex molecules interact with each other to form even larger assembled structures. The findings of this project are likely to influence how we think about living systems. This project is the vehicle for advanced training of two graduate students and two undergraduate researchers. In addition to formal training activities, the Conboy and Babst research groups are participating in public outreach activities to bring science to a wider audience.Our current view of the cell membrane was established in the early 1970’s by Singer and Nicolson. The fluid-mosaic model they proposed portrays the membrane as a “liquid-like” bilayer of lipids, cholesterol, and proteins, exhibiting rapid and free diffusion in the two dimensions parallel to the membrane surfaces. In stark contrast, the exchange of lipids between the leaflets of a bilayer was presumed to be prohibited by the large energetic barrier associated with translocating the hydrophilic lipid headgroup, through the hydrophobic membrane core. This static picture of lipid translocation (or flip-flop) has been a long-held belief in the study of membrane dynamics and is the basis for the current theories regarding the bilateral organization of cell membrane components, particularly phosphatidylserine (PS) asymmetry. We hypothesize that spontaneous PS flip-flop is a common, facile process in vivo. This prediction raises the question: How does the cell maintain PS asymmetry in the plasma membrane? Our hypothesis is that in vivo PS localization to the cytosolic leaflet of the membrane is driven by electrostatic interactions with cytoplasmic proteins, in particular, the proteins comprising the cytoskeleton. Our proposed model of PS asymmetry and the currently accepted view differ in that our model suggests a very dynamic membrane system, over the more conventional “static” picture of lipid asymmetry. This dynamic behavior changes the current theories regarding the creation and maintenance of lipid asymmetry; however, it also simplifies and unifies the explanation of many known lipid phenomena. Our hypothesis is being tested by construction of a unique sum-frequency vibrational microscope capable of directly quantifying and imaging lipid asymmetry in living cells and measuring native lipid flip-flop in vivo for the first time. The students engaged in this project are gaining knowledge and experience in rigorous physical and biophysical concepts as well as advanced imaging techniques based on nonlinear optical processes. Outreach activities include engagement of K-12 schools in the Salt Lake Valley, especially Catholic schools that serve higher percentages of underrepresented groups.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.