Probing physical limits in TRPV1-magnetogenetics with micro-magnetic devices

离子通道是控制穿过细胞膜的离子流动的门。离子通道的激活(开放)触发了细胞和组织中关键信号过程的级联。大多数离子通道是对化学或电刺激做出反应而激活的，但也有一些称为机械敏感通道的离子通道专门对机械压力(如拉伸或剪切应力)做出反应。“磁遗传学”是一种利用磁性远程非侵入性地控制机械敏感通道来影响基因表达的方法。该项目将设计一种磁系统，以放大并在空间上瞄准磁致反应。磁性纳米棒或铁隔离蛋白将附着在机械敏感通道上，并将应用工程磁场来操纵通道行为。这种新的离子通道控制系统将被用来研究机械敏感通道对一定范围的力的响应。这项研究有望对机械敏感离子通道的生物物理学产生新的理解，并为如何远程靶向或激活此类通道提供新的策略。所开发的方法可能应用于依赖于机械信号的各种生物过程。该教育计划的重点是在年轻学生(通常是代表性不足的学生)中传授广泛的技能，包括设计、制造、模拟和生物技术。研究人员试图通过向年轻学生介绍从工程设计和制造到研究和分析生物系统响应的综合项目，来创造对STEM的长期兴趣。现代生物医学工程的一个核心目标仍然是开发能够接触和控制生命系统底层机械的工具。该项目将开发集成的磁性设备，以空间控制和放大TRPV1-磁性发生细胞的生物磁反应。这些工具将首先被用来全面地表征磁发生钙信号。这将通过以下方式实现：(1)将微磁放大器设备连接到这样的单元(其行为类似于大规模平行的、可调的磁陷阱)，以及(2)改变标记在TRPV1上的纳米磁铁的磁容量。磁系统将产生在磁力发电系统中产生的最高静态或时变磁场/磁场梯度，并将产生超过打开TRPV1通道所需的理论力的通道标记机械力。此外，详细研究静态或时变的磁场、磁场梯度、拉力速率和纳米磁体体积如何在2D空间中相互作用来极化细胞内的钙信号，将有助于阐明(或驳斥)磁发生行为。该项目将解决关于磁生成学的可行性和背后的物理机制的新出现的争议。操纵离子通道的磁性方法具有巨大的潜力，使非侵入性工具能够控制生物行为。对该项目的调查对于阐明磁生控制的限度和澄清如何实际利用这种战略是至关重要的一步。使用钙成像、膜片钳和单细胞成像/探测相结合的现代技术，在体外将在单细胞和大规模平行尺度上对细胞进行全面探测。由于TRPV1离子通道在神经系统中含量丰富，并在许多功能中发挥重要作用，预计这些系统将为高度局部化刺激下机械敏感通道的物理学提供新的见解，并实现体外神经工程的新平台。由于在数据收集、蛋白质靶向和机械力产生方面的可扩展性，这些工具可能还会发现对各种蛋白质的机械传导性质的新见解。这一奖项反映了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。

Ion channels are gates that control the flow of ions across the cell membrane. Activation (opening) of ion channels triggers cascades of critical signaling processes in cells and tissues. Most ion channels are activated in response to chemical or electrical stimuli, though some called mechanosensitive channels specifically respond to mechanical forces such as stretch or shear stress. "Magnetogenetics" is a method that uses magnetism to remotely and noninvasively control mechanosensitive channels to affect gene expression. This project will engineer a magnetic system to amplify and spatially target magnetogenetic responses. Magnetic nanobeads or iron-sequestering proteins will be attached to the mechanosensitive channels, and engineered magnetic fields will be applied to manipulate channel behavior. This new system of ion channel control will be used to study the response of mechanosensitive channels to a range of force. The research is expected to yield new understanding on the biophysics of mechanosensitive ion channels and inform new strategies on how to target or activate such channels remotely. The methods developed could potentially be applied to a variety of biological processes that depend on mechanical signals. The education program is focused on teaching broad skills among younger (and often underrepresented) students, encompassing design, fabrication, simulation, and biotechnology. The investigators seek to create long-term interest in STEM by introducing younger students to integrative projects that link from design and fabrication in engineering, to studying and analyzing the response of biological systems. A core goal of modern biomedical engineering remains the development of tools that can engage and control the underlying machinery of living systems. This project will develop integrated magnetic devices to spatially-control and amplify biomagnetic response in TRPV1-magnetogenetic cells. These tools will first be used to comprehensively characterize magnetogenetic Ca2+ signaling. This will be accomplished by: (1) Interfacing micro-magnetic amplifier devices to such cells (that would behave like massively-parallel, tunable magnetic traps), and (2) Varying the magnetic volume of nano-magnets tagged to TRPV1. The magnetic systems will create the highest static or time-varying magnetic field / magnetic-field gradients ever elicited in magnetogenetic systems, and will generate channel-tagged mechanical forces that exceed the theoretical force required to open TRPV1 channels. In addition, detailed studies on how static or time-varying magnetic field, field gradient, pull-rate, and nano-magnet volume interact over 2D space to polarize cellular Ca2+ signaling will help clarify (or refute) magnetogenetic behavior. The project will address emerging controversy on the viability of, and physical mechanisms behind magnetogenetics. Magnetic approaches to manipulate ion channels carry tremendous, as of yet unrealized potential to enable non-invasive tools to control biological behavior. The investigations of this project are a critically important step to both laying out the limits of magnetogenetic control and clarifying how such strategies may be practically utilized. Cells will be comprehensively probed at both the single cell and massively-parallel scales in-vitro, using modern techniques in calcium imaging, patch-clamping, and combined single-cell imaging/probing. It is anticipated that these systems will yield new insights on the physics of mechanosensitive channels under highly-localized stimuli and realize a new platform for in-vitro neuroengineering, as TRPV1 ion channels are abundant in the nervous system and play an important role in many functions, e.g., the regulation of pain. Such tools may additionally uncover new insights on the mechanotransductive nature of a wide variety of proteins due to their scalability in data collection, protein targeting, and mechanical force generation.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.