Nanoscale probes for sensing molecular functions in live cells

项目摘要/摘要： 该Mira提案合并了由R01GM128142支持的两个不同的项目，即膜的作用 表面纳米拓扑术诱导的细胞功能中的曲率“，以及R01GM125737，”发展中的纳米尺度“ 用于强健细胞内记录的电生理传感器“。虽然这两个项目关注的是不同的生物 问题，统一的主题是开发纳米级的探测器来阐明复杂的 活细胞的环境。在这项提案中，我们按照家长拨款的思路来讨论主题，重点是 生物学问题的重要性，我们最近和不断发展的结果，以及未来的方向。为 第一个项目的长期目标是了解细胞膜曲率如何调节生化信号 它们通过单元-矩阵接口传输。在细胞-矩阵交界处，细胞在那里产生物理 与细胞外基质接触时，膜可能会因基质形貌或 机械力。由于在活细胞中操纵纳米尺度的膜曲率仍然是一个挑战，我们的 目前对局部膜曲率如何影响信号传输的了解有限。我们建议 利用基于纳米技术的精密工程来控制活细胞中的界面膜曲率。我们 试图了解细胞膜曲率和潜在的分子是如何影响细胞过程的 机械装置。所获得的知识将有助于我们理解细胞如何与细胞外基质和 还可以帮助我们设计生物材料，以便更好地与细胞整合。对于第二个项目，我们正在开发 将垂直纳米电极转化为坚固易用的电生理工具，可可靠地实现并联 最小扰动下的心肌细胞内记录。同时使用纳米电极和贴片 在相同细胞上的钳制记录证实，纳米电极准确地记录了动作电位波形 用于干细胞来源的心肌细胞的分类和表征。这些纳米电极将使 我们希望了解体外干预如何促进干细胞来源的心肌细胞成熟。 此外，纳米电极为监测膜的生成和再密封提供了理想的工具 心肌细胞上的孔，由于其大小和强大的机械性能而容易膜破裂 收缩。我们将使用纳米电极来研究蛋白质如何参与膜的再密封 进程。我们希望通过将新工具的开发与应用程序相结合来实现广泛的影响 特定的生物系统。

Project Summary / Abstract: This MIRA proposal merges two distinct projects supported by R01GM128142, “The role of membrane curvature in surface nanotopography-induced cell functions”, and R01GM125737, “Developing nanoscale electrophysiology sensors for robust intracellular recording”. While the two projects focus on different biological questions, the unifying theme is to develop nanoscale probes to elucidate the cellular machinery in the intricate environment of living cells. In this proposal, we discuss topics along the lines of the parent grants, focusing on the significance of the biological problems, our recent and evolving results, and directions for the future. For the first project, the long-term goal is to understand how membrane curvature regulates biochemical signals that are transmitted through the cell-matrix interface. At the cell-matrix interface, where the cells make physical contact with extracellular matrices, the membrane may be locally deformed by matrix topography or mechanical forces. As it remains a challenge to manipulate nanoscale membrane curvature in live cells, our current understanding of how local membrane curvature affects signal transmission is limited. We propose to use nanotechnology-based precision engineering to control interface membrane curvature in live cells. We seek to understand how cellular processes are affected by membrane curvature and the underlying molecular mechanisms. The knowledge gained will help us understanding how cells interact with extracellular matrix and also help us designing biomaterials for better integration with cells. For the second project, we are developing vertical nanoelectrodes into a robust and easy-to-use electrophysiology tool that can reliably achieve parallel intracellular recording of cardiomyocytes with minimal perturbation. Simultaneous nanoelectrode and patch clamp recordings on same cells confirmed that nanoelectrodes accurately record action potential waveforms for classification and characterization of stem-cell-derived cardiomyocytes. These nanoelectrodes will enable us to understand how in vitro interventions accelerate the maturation of stem-cell-derived cardiomyocyte. Furthermore, nanoelectrodes provide an ideal tool for monitoring the generation and resealing of membrane pores on cardiomyocytes that are prone to membrane rupture due to their large size and strong mechanical contraction. We will use nanoelectrode to investigate how proteins participate in the membrane resealing process. We hope to achieve a broad impact by combining the development of new tools with applications to specific biological systems.