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

离子通道是控制离子穿过细胞膜流动的门。 离子通道的激活（开放）触发细胞和组织中关键信号传导过程的级联。大多数离子通道响应于化学或电刺激而被激活，尽管一些被称为机械敏感通道的通道特异性地响应于机械力，例如拉伸或剪切应力。“磁遗传学”是一种利用磁性远程和非侵入性地控制机械敏感通道以影响基因表达的方法。该项目将设计一个磁系统来放大和空间靶向磁致反应。磁性纳米珠或铁螯合蛋白将附着在机械敏感通道上，并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用于研究机械敏感通道对一系列力的响应。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。所开发的方法可以潜在地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（而且往往代表性不足）的学生中教授广泛的技能，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反应的综合项目，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参与和控制生命系统底层机制的工具。该项目将开发集成磁性设备，以空间控制和放大TRPV 1-磁发生细胞中的生物磁响应。这些工具将首先用于全面表征磁致Ca 2+信号。这将通过以下方式实现：（1）将微磁放大器设备与这些细胞连接（表现得像平行的可调磁阱），以及（2）改变标记到TRPV 1的纳米磁体的磁体积。磁系统将产生磁发生系统中有史以来引起的最高静态或时变磁场/磁场梯度，并将产生超过打开TRPV 1通道所需的理论力的通道标记机械力。此外，关于静态或时变磁场、场梯度、拉速和纳米磁体体积如何在二维空间上相互作用以抑制细胞Ca 2+信号传导的详细研究将有助于澄清（或反驳）磁发生行为。该项目将解决磁遗传学的可行性和背后的物理机制的新出现的争议。 磁方法来操纵离子通道进行巨大的，尚未实现的潜力，使非侵入性工具来控制生物行为。这个项目的调查是一个至关重要的一步，既奠定了磁致控制的限制，并澄清如何这种策略可能实际使用。细胞将在体外以单细胞和平行的尺度进行全面探测，使用钙成像、膜片钳和组合的单细胞成像/探测中的现代技术。预计这些系统将对高度局部化刺激下的机械敏感通道的物理学产生新的见解，并实现体外神经工程的新平台，因为TRPV 1离子通道在神经系统中丰富并且在许多功能中发挥重要作用，例如，疼痛的调节 这些工具还可以发现各种蛋白质的机械转导性质的新见解，因为它们在数据收集，蛋白质靶向和机械力产生方面具有可扩展性。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.