Optical Control of Protein Activity in Live Cells by Plasmon Assisted Light Inactivation

摘要 光学工具具有无与伦比的空间和时间精度，有助于更好地理解 现代医学和生物学中的各种过程。母公司R35奖专注于开发工具，以 基于等离子体纳米粒子脉冲激光加热的活细胞中蛋白质活性的光学控制及其 热限制加热以在几纳米范围内展开和变性周围的蛋白质 纳米颗粒表面。母体R35的重点是两个方面：(1)更好地理解激光-纳米材料 相互作用，包括纳米颗粒附近的纳米级温度和生化 受影响的蛋白质的反应；以及(2)开发这种新的光学工具来操纵活体中的蛋白质活性 细胞，侧重于G蛋白偶联受体(GPCR)，这是一类重要的和多样化的膜 介导细胞外信号到细胞内信号的受体。我们最近的研究表明，更短的时间 皮秒激光刺激导致金的光声响应显著增强 由于电子-声子(e-ph)的直接耦合，包覆了一层薄二氧化硅(Au@SiO_2)的纳米颗粒 通过金-硅胶界面，增强了硅胶-水界面的界面热传递。我们进一步 发现内皮靶向金纳米颗粒(AuNPs)的皮秒激光激发产生了 纳米尺度的机械扰动，或光声效应，并激活机械敏感离子通道 (TRPV4，Piezo1)和G蛋白偶联受体在活细胞中。我们希望进一步继续这些 通过阐明机制和建立生物医学技术来远程控制 受体和细胞活性与光学分辨率。这项拟议的补编要求综合两项- 光子刺激和成像系统使这些努力成为可能。科学上的理由就是这么短 飞秒激光刺激产生了比皮秒或纳秒更强的非平衡 激光刺激，并产生纳米级的机械/声学响应，以光学控制受体和细胞 活动。我们目前正在进行的研究将受益于这种集成的刺激和成像系统，因为 目前使用的激光器可以集成到系统的光路中，并利用实时 成像能力。这项拟议的补充设备符合母公司授予的范围，因为它将 提供一种系统，以(1)通过激光-纳米材料相互作用获得更广泛的时间尺度；(2)提供光学 允许实时刺激和成像其自然环境中的细胞活动的系统；(3)调查 包括飞秒激光在内的脉冲激光如何控制蛋白质活性和细胞反应。所有这些都是 这些努力需要综合的刺激和成像。因此，所要求的补充材料提供了一个令人兴奋的 研究激光-纳米材料相互作用的基本机制并建立生物医学的机会 光学控制受体和细胞活动的技术。

Abstract Optical tools have unparalleled spatial and temporal precision and have been instrumental to better understand various processes in modern medicine and biology. The parent R35 award focuses on developing tools for optical control of protein activity in live cells, based on pulsed laser heating of plasmonic nanoparticles, and its thermally confined heating to unfold and denature surrounding proteins within a few nanometers of nanoparticle surface. The focus of the parent R35 is two-fold: (1) better understand the laser-nanomaterial interactions including the nanoscale temperature in the proximity of the nanoparticle and biochemical responses of the affected proteins; and (2) developing this new optical tool to manipulate protein activity in live cells with emphasis on G-protein coupled receptors (GPCR), an important and diverse class of membrane receptors that mediate extracellular to intracellular signaling. Our recent studies have revealed that shorter picosecond laser stimulation leads to a significant enhancement of the photoacoustic response for gold nanoparticles coated with a thin shell of silica (Au@SiO2), due to the direct electron-phonon (e-ph) coupling across the gold-silica interface and enhanced interfacial heat transfer at the silica-water interface. We further discovered that the picosecond laser excitation of endothelial-targeted gold nanoparticles (AuNPs) generates a nanoscale mechanical perturbation, or photoacoustic effect, and activates mechanosensitive ion channels (TRPV4, Piezo1) and G-protein coupled receptors in live cells. We would like to further continue these investigations by elucidating the mechanism and building biomedical technologies to remotely control the receptor and cell activities with optical resolution. This proposed supplement requests an integrated two- photon stimulation and imaging system to enable these efforts. The scientific rationale is that shorter femtosecond laser stimulation creates a stronger non-equilibrium than even with picosecond or nanosecond laser stimulation, and generates nanoscale mechanical/acoustic response to optically control receptor and cell activities. Our current ongoing studies would benefit from this integrated stimulation and imaging system, as the currently used lasers can be integrated into the optical pathways of the system and utilize the real-time imaging capability. This proposed supplemental equipment fits the scope of the parent award since it would provide a system to (1) access broader timescales by laser-nanomaterial interactions; (2) provide an optical system to allow real-time stimulation and imaging of cellular activities in its native environment; (3) investigate how the pulsed laser including femtosecond laser can control protein activity and cellular responses. All these efforts require integrated stimulation and imaging. Therefore the requested supplement provides an exciting opportunity to investigate the fundamental mechanism of laser-nanomaterial interactions and build biomedical technologies for optical control of the receptor and cellular activities.