Engineering photostable fluorescent proteins and biosensors using transcriptomic mining and massive-throughput single-cell screening

使用转录组挖掘和大通量单细胞筛选来工程光稳定荧光蛋白和生物传感器

• 批准号: 10610472
• 负责人: Francois St-Pierre
• 金额: $ 60.14万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10610472
• 关键词: Address; Adopted; Agar; Aging; Attention; Benchmarking; Biological; Biological Assay; Biomedical Research; Biophysics; Biosensor; Bulla; Calcium; Cell Cycle; Cell Death; Cell Shape; Cell physiology; Cells; Color; Coupling; Cytometry; Dedications; Detection; Development; Disease; Electrophysiology (science); Embryo; Engineering; Equipment; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Fluorescent Probes; Functional disorder; Gene Expression; Gene Proteins; Genetic Engineering; Health; Image; Imaging Device; Individual; Jellyfish; Label; Laboratories; Lasers; Learning; Light; Lighting; Mammalian Cell; Marine Invertebrates; Medicine; Membrane; Metagenomics; Methods; Microscopy; Mining; Modeling; Morphology; Mutagenesis; Neurons; Nuclear; Optics; Pattern; Photobleaching; Photons; Procedures; Productivity; Property; Protein Engineering; Proteins; Protocols documentation; Rapid screening; Reagent; Recovery; Reporter; Reporter Genes; Reporting; Research Proposals; Resolution; Resource Sharing; Sampling; Science; Signal Transduction; Site-Directed Mutagenesis; Specificity; Structure; Syncope; System; Techniques; Technology; Tissue imaging; Variant; cell type; cellular imaging; chromophore; cofactor; college; design; experimental study; fluorophore; high throughput screening; imaging modality; imaging probe; improved; interest; invention; metagenome; millisecond; monomer; neuroimaging; new technology; novel; nucleic acid localization; red fluorescent protein; screening; single molecule; temporal measurement; transcriptome; transcriptomics; two-photon; voltage

PROJECT SUMMARY/ABSTRACT Fluorescent proteins are ubiquitous reagents in the biomedical sciences for reporting gene expression, protein and nucleic acid localization, cell shape, and cellular activity. However, fluorescent proteins (FPs) become progressively dimmer — they photobleach — with repeated or prolonged illumination. Photobleaching limits multiple types of biological experiments where photostability is essential, such as single-molecule biophysics and timelapse imaging of cellular activity during development, learning, and aging. Photobleaching often cannot simply be addressed by increasing the excitation light, as high illumination power can induce membrane blebbing, nuclear fragmentation, alterations in the cell cycle, changes to the concentration of intracellular calcium, and, ultimately, cell death. While over two decades of FP engineering has led to a toolbox of bright FPs, less attention has been devoted to improving photostability because of the greater difficulty and lower throughput endured when screening for photostable FPs. Moreover, few studies have attempted to improve photophysical properties under two-photon illumination — a method of choice for deep-tissue imaging — because of technical challenges associated with screening under this imaging modality. The overall objective of this research proposal is, therefore, to develop and apply a color palette of bright and photostable FPs for one- and two-photon imaging in mammalian cells. Our proposal leverages two specialized and synergistic approaches to FP discovery and engineering: (1) SPOTlight, a new all- optical screening approach developed in Dr. St-Pierre's lab that circumvents technical hurdles and enables rapid screening of both brightness and photostability at the single-cell level under one- and two-photon illumination; and (2) transcriptomic and metagenomic mining for novel FPs from marine invertebrates, a technique pioneered by Dr. Shaner’s lab. SPOTlight relies on light patterning technology to selectively illuminate individual cells labeled with fluorophores that can be photoactivated from a dim to a bright state. The cells are therefore tagged with a unique fluorescence signature that can then be distinguished and retrieved using Fluorescence Activated Cell Sorting (FACS). SPOTlight thus enables screening in dense mixed cultures with single-cell resolution, thereby eclipsing the throughput of traditional well-based approaches. Mining for novel FPs in marine invertebrate transcriptomes and metagenomes will allow us to rapidly identify and characterize hundreds of novel FPs. From this pool of new FPs, we will select the most photostable for engineering with the SPOTlight pipeline. We will also model their structures to guide site-directed mutagenesis. We propose to leverage these new technologies and assays to develop FPs of different colors that are bright, monomeric, and sufficiently photostable for long-term imaging experiments. We also propose to apply these new FPs to increase the photostability of genetically encoded voltage indicators (GEVIs), which are fluorescent biosensors whose brightness reports changes in voltage. While GEVIs are proposing tools for imaging neural electrical activity with exquisite temporal resolution, they require high illumination power for detection and typically bleach in seconds or minutes. Overall, we anticipate that this project will produce bright and photostable fluorophores and biosensors of broad utility for illuminating cellular dynamics and that our procedures will inspire further multi-parameter engineering of imaging probes for long-term imaging.

项目总结/摘要 荧光蛋白是生物医学科学中普遍存在的用于报告基因表达、蛋白质和核酸的试剂。 酸定位、细胞形状和细胞活性。然而，荧光蛋白（FP）变得越来越暗- 它们通过反复或长时间的照射而光漂白。光漂白限制了多种类型的生物实验 其中光稳定性是必不可少的，例如单分子生物物理学和细胞活动的时移成像， 发育、学习和衰老。光漂白通常不能简单地通过增加激发光来解决， 高光照功率可诱导膜起泡、核碎裂、细胞周期的改变、以及细胞周期的改变。 细胞内钙浓度，最终导致细胞死亡。虽然二十多年的FP工程已经导致了一个 在明亮的FP工具箱中，较少关注改善光稳定性，因为其难度更大， 在筛选光稳定FP时，通量持续存在。此外，很少有研究试图改善生物物理 由于技术挑战，双光子照明下的性能-深层组织成像的首选方法 与这种成像模式下的筛查相关。因此，本研究提案的总体目标是： 开发并应用明亮且耐光的FP调色板，用于哺乳动物细胞中的单光子和双光子成像。我们 建议利用两个专门的和协同的方法来FP发现和工程：（1）SPOTlight，一个新的全- 在圣皮埃尔博士的实验室开发的光学筛选方法，绕过技术障碍，使快速筛选 在单光子和双光子照射下，在单细胞水平上的亮度和光稳定性;和（2）转录组学 以及从海洋无脊椎动物中挖掘新型FP的宏基因组学，这是沙纳博士实验室开创的一项技术。聚光灯 依赖于光图案化技术来选择性地照射用荧光团标记的单个细胞， 光活化从暗淡到明亮的状态。因此，这些细胞被标记上独特的荧光标记， 使用荧光激活细胞分选（FACS）来区分和检索。因此，SPOTlight可以在 高密度混合培养物具有单细胞分辨率，从而使传统的基于孔的方法的通量黯然失色。 在海洋无脊椎动物转录组和宏基因组中挖掘新的FP将使我们能够快速识别和 描述了数百种新颖的FP。从这个新的FP池中，我们将选择最耐光的工程， SPOTlight管道。我们还将模拟它们的结构来指导定点诱变。我们建议利用 这些新的技术和分析方法可以开发出不同颜色的FP，这些FP是明亮的，单体的，并且足够耐光 用于长期成像实验。我们还建议应用这些新的FP来增加遗传修饰的光稳定性。 编码电压指示器（GEVI），这是荧光生物传感器，其亮度报告电压的变化。而 GEVI正在提出具有精致时间分辨率的神经电活动成像工具，它们需要高分辨率 用于检测的照明功率并且通常在数秒或数分钟内漂白。总的来说，我们预计该项目将 产生明亮且光稳定的荧光团和生物传感器，其广泛用于照亮细胞动力学， 这些过程将激发用于长期成像的成像探针的进一步多参数工程。