Expanding the HIDE nanoscopy toolbox: More organelles, colors, and modalities

扩展 HIDE 纳米镜工具箱：更多细胞器、颜色和模式

• 批准号: 10019809
• 负责人: Alanna Schepartz
• 金额: $ 26.19万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10019809
• 关键词: 3-Dimensional; Address; Cell membrane; Cells; Cellular biology; Chemicals; Chemistry; Cilia; Color; Complement; Complex; Coupled; Cyclooctenes; Disease; Dyes; Electron Microscopy; Endosomes; Ensure; Environment; Foundations; Goals; Golgi Apparatus; Health; Hydrophobicity; Image; In Situ; Ions; Label; Ligation; Lipids; Membrane; Methods; Microscopy; Minor; Modality; Nanoscopy; Organelles; Proteins; Protocols documentation; Reaction; Recycling; Regulation; Reporting; Research; Resolution; Rhodamine; Scanning Electron Microscopy; Scientist; Silicon; Structure; Technology; Time; Validation; Work; cell growth regulation; density; design; fluorophore; functional group; imaging modality; improved; innovation; microscopic imaging; mitochondrial membrane; nanometer; novel; preservation; single molecule; tool; virtual

Project Summary: A major goal of cell biology is to understand the structures, interactions, functions and regulation of cellular organelles. Today, Nobel-worthy research of Betzig, Hell, Moerner, Zhuang, and now many others allows scientists to visualize organelles in live cells at resolutions that number in the tens of nanometers. The problem is that the images last for only seconds, and thus the pictures, however beautiful, hide complex dynamics and interactions that underlie organelle function in health and disease. This proposal expands a toolkit of two-component chemical tools called ‘HIDE’ (High Density Environment-Sensitive) probes that will allow scientists to visualize the dynamics of multiple organelles, simultaneously, at super-resolution, for unprecedented times, and in multiple colors. HIDE probes consist of two parts, an organelle- specific membrane probe with a reactive functional group (such as trans-cyclooctene (TCO)) and a silicon rhodamine dye (SiR or, in this proposal, HMSiR) with a reaction partner (such as tetrazine). These two parts, when added sequentially to live cells, undergo an in situ tetrazine ligation reaction that localizes the SiR dye at high density within the organelle membrane. Here HMSiR photostability is significantly enhanced and can generate single molecule switching (SMS) nanoscopy images up to 50 times longer than HMSiR-tagged proteins. The first HIDE probes (used to image the Golgi, ER, mitochondria, and plasma membrane) have been reported by us. Here we build on this strong foundation and expand the HIDE toolbox to include probes for four additional organelles (early, late, and recycling endosomes, and cilia) (Aim 1), coupled with orthogonal SMS dyes and chemistries (Aim 2) required for two- and eventually three-color long-term SMS nanoscopy. In the final Aim (Aim 3), we develop HIDE-EM probes to highlight discrete organelles in 3D by electron microscopy (EM). Each Aim is associated with deliverables and time-dependent milestones and is supported by significant preliminary results. Although we focus here on developing HIDE probes useful for SMS microscopy, only minor adjustments in the dye identity would be needed to generate HIDE probes for STED. The technology developed herein will greatly enhance the power, scope, and impact of long time-lapse live cell nanoscopy by expanding the number and types of organelles that can be evaluated, alone, in combination using multiple colors, using different imaging modalities, and in both cultured and hard-to-transfect primary cells.

项目概要： 细胞生物学的一个主要目标是了解结构、相互作用、功能和调控 的细胞器。今天，贝齐格、赫尔、莫尔纳、庄的诺贝尔奖研究，以及现在 许多其他技术使科学家能够以数以万计的分辨率可视化活细胞中的细胞器 几十纳米。问题是图像只持续几秒钟，因此 图片无论多么美丽，都隐藏着细胞器背后复杂的动态和相互作用 在健康和疾病中发挥作用。该提案扩展了双组分化学品的工具包 称为“HIDE”（高密度环境敏感）探针的工具将使科学家能够 同时以超分辨率可视化多个细胞器的动态 前所未有的时代，并且有多种颜色。 HIDE 探针由两部分组成，一个是细胞器- 具有反应性官能团（例如反式环辛烯（TCO））的特定膜探针 以及硅罗丹明染料（SiR，或在本提案中为 HMSiR）与反应伙伴（例如 四嗪）。当这两个部分依次添加到活细胞中时，会发生原位四嗪 连接反应将 SiR 染料以高密度定位在细胞器膜内。这里 HMSiR光稳定性显着增强，可产生单分子开关 (SMS) 纳米显微镜图像比 HMSiR 标记蛋白质长 50 倍。第一个隐藏 探针（用于对高尔基体、内质网、线粒体和质膜进行成像）已被报道 由我们。在这里，我们建立在这个坚实的基础上，并扩展 HIDE 工具箱以包括探针 对于四个额外的细胞器（早期、晚期、回收内体和纤毛）（目标 1），耦合 使用两色和最终三色所需的正交 SMS 染料和化学品（目标 2） 长期 SMS 纳米镜检查。在最终目标（目标 3）中，我们开发了 HIDE-EM 探针来突出显示 通过电子显微镜 (EM) 观察 3D 离散细胞器。每个目标都与可交付成果相关联 和时间相关的里程碑，并得到重要初步结果的支持。虽然我们 这里的重点是开发可用于 SMS 显微镜的 HIDE 探针，仅进行细微调整 需要染料身份来生成 STED 的 HIDE 探针。技术 本文开发的技术将大大增强长时间延时活细胞的能力、范围和影响 纳米显微镜通过扩大可以单独评估的细胞器的数量和类型 使用多种颜色、使用不同成像方式的组合，以及在培养和 难以转染的原代细胞。

项目成果

Imaging organelle membranes in live cells at the nanoscale with lipid-based fluorescent probes.

• DOI: 10.1016/j.cbpa.2021.09.003
• 发表时间: 2021-12
• 期刊: Current opinion in chemical biology
• 影响因子: 7.8

Long-Term Live-Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI-SiR.

与质膜隐藏探针DII-SIR的原代和培养细胞的长期活细胞纳米镜检查。

• DOI: 10.1002/anie.201704783
• 发表时间: 2017-08-21
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Thompson AD;Omar MH;Rivera-Molina F;Xi Z;Koleske AJ;Toomre DK;Schepartz A
• 通讯作者: Schepartz A

Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles.

生物正交的，电压敏感荧光团的荧光靶向，以可视化细胞细胞器中的膜电位动力学。

• DOI: 10.1021/jacs.2c02664
• 发表时间: 2022-07-13
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Klier PEZ;Gest AMM;Martin JG;Roo R;Navarro MX;Lesiak L;Deal PE;Dadina N;Tyson J;Schepartz A;Miller EW
• 通讯作者: Miller EW