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

项目摘要/摘要 荧光蛋白是生物医学科学中普遍存在的报告基因表达、蛋白质和核的试剂 酸定位、细胞形状和细胞活性。然而，荧光蛋白(FP)逐渐变得更暗- 他们用重复或长时间的光照来漂白。光漂白限制了多种类型的生物实验 光稳定性至关重要的地方，如单分子生物物理学和细胞活动的时间流逝成像 发展、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，因为 高强度光照可诱导细胞膜起泡、核碎裂、细胞周期改变等。 细胞内钙离子浓度，最终导致细胞死亡。虽然二十多年的FP工程已经导致了 由于较大的难度和较低的成本，对提高光稳定性的关注较少 筛选光稳定的FP时所承受的吞吐量。此外，很少有研究试图改进光物理 由于技术挑战，双光子照明下的特性--深层组织成像的一种选择方法-- 与这种成像模式下的筛查有关。因此，这项研究提案的总体目标是 开发和应用一种明亮和光稳定的FP调色板，用于哺乳动物细胞的单光子和双光子成像。我们的 Proposal利用两种专门的和协同的方法来进行FP发现和工程：(1)聚光灯，一种新的全新的 圣皮埃尔博士实验室开发的光学筛查方法绕过了技术障碍，实现了快速筛查 在单细胞水平上在单光子和双光子照射下的亮度和光稳定性；以及(2)转录 以及从海洋无脊椎动物中挖掘新的FP的元基因组学，这是沙纳博士的实验室首创的一项技术。聚光灯 依靠光图案化技术选择性地照亮标有荧光团的单个细胞，这些荧光团可以 从昏暗到明亮的光激活状态。因此，细胞被标记了独特的荧光信号，然后可以 使用荧光激活细胞分选(FACS)进行区分和检索。因此，Spotlight可以在 具有单细胞分辨率的密集混合培养，从而使传统的基于良好的方法的吞吐量黯然失色。 挖掘海洋无脊椎动物转录本和后基因组中的新FP将使我们能够快速识别和 描述数以百计的新奇FP。从这个新的FP池中，我们将选择最稳定的照片来进行工程设计 聚光灯管道。我们还将对它们的结构进行建模，以指导定点突变。我们建议利用 这些新技术和分析方法旨在开发不同颜色的FP，这些FP是明亮的、单体的和足够光稳定的 用于长期成像实验。我们还建议应用这些新的FP来提高基因的光稳定性 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。而当 GEVI正在提出具有精细时间分辨率的神经电活动成像工具，它们需要很高的 用于检测的照明功率，通常在几秒钟或几分钟内漂白。总体而言，我们预计这个项目将 生产明亮和光稳定的荧光团和生物传感器，广泛用于照亮细胞动力学，并且我们的 该程序将启发进一步的多参数工程的成像探头，以长期成像。

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.