mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging.
mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging.
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DOI：
10.1073/pnas.2000942117
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发表时间：
2020-12-01
影响因子：
11.1
通讯作者：
Petsko GA
中科院分区：
文献类型：
--
作者：
Campbell BC;Nabel EM;Murdock MH;Lao-Peregrin C;Tsoulfas P;Blackmore MG;Lee FS;Liston C;Morishita H;Petsko GA
我们已经开发了一种荧光蛋白，mGreenLantern，其在小鼠，细菌和人类细胞中具有非常高的亮度（比EGFP亮六倍），并已证明其上级能力，突出显示神经元形态相比，EGFP和EYFP。基于全细胞亮度筛选荧光蛋白突变体，同时评估裂解物中的表达动力学，使我们能够鉴定出在其计算的光谱亮度和细胞中的实际性能之间表现出惊人差异的变体。mGreenLantern还具有异常高的化学和热力学稳定性，并与现有的GFP过滤器组，激发源，商业EGFP抗体，扩增显微镜和全脑组织清除兼容。我们的假设驱动的工程策略代表了一种具有巨大潜力的可推广的方法，以提高组成型报告和基于GFP的生物传感器的性能。尽管增强型绿色和黄色荧光蛋白（EGFP和EYFP）在生物学研究中无处不在，但它们并没有特别针对神经科学进行优化，而且它们在脑组织中的亮度和缓慢表达限制了树突棘分析和其他研究神经发育和可塑性不可或缺的技术的保真度。我们假设，EGFP在哺乳动物系统中的低溶解度必须限制整个细胞的总荧光输出，因此提高折叠效率可以转化为表达神经元的更大亮度。通过将合理选择的折叠增强突变组合引入GFP模板并筛选人类细胞中的亮度和表达率，我们开发了mGreenLantern，一种在细胞中具有比EGFP高六倍亮度的荧光蛋白。mGreenLantern可在72小时内照亮小鼠大脑中的神经元，大大缩短了病毒转导和成像之间的滞后时间，同时其高亮度可改善使用宽视野、共聚焦和双光子显微镜对神经元形态的检测。当病毒表达到体内投射神经元时，mGreenLantern荧光的发展速度比EYFP快四倍，并突出显示了在EYFP标记的细胞中难以检测到的长程过程。此外，mGreenLantern在组织清除和扩张显微镜检查后保留了强荧光，从而促进了超分辨率和全脑成像，而无需免疫组织化学。mGreenLantern可以在不同的系统中直接取代EGFP/EYFP，因为它与GFP过滤器组兼容，被EGFP抗体识别，并且在小鼠，人类和细菌细胞中表现出色。我们的筛选和合理的工程方法是广泛适用的，并表明荧光蛋白，包括生物传感器，可以使用类似的策略解锁更大的潜力。
We have developed a fluorescent protein, mGreenLantern, that features exceptionally high brightness in mouse, bacterial, and human cells (up to sixfold brighter than EGFP) and have demonstrated its superior ability to highlight neuronal morphology compared to EGFP and EYFP. Screening fluorescent protein mutants based on whole-cell brightness while evaluating expression kinetics in lysate enabled us to identify variants exhibiting striking divergences between their computed spectroscopic brightness and actual performance in cells. mGreenLantern additionally features unusually high chemical and thermodynamic stability and is compatible with existing GFP filter sets, excitation sources, commercial EGFP antibodies, expansion microscopy, and whole-brain tissue clearing. Our hypothesis-driven engineering strategy represents a generalizable method with great potential to enhance the performance of constitutive reporters and GFP-based biosensors. Although ubiquitous in biological studies, the enhanced green and yellow fluorescent proteins (EGFP and EYFP) were not specifically optimized for neuroscience, and their underwhelming brightness and slow expression in brain tissue limits the fidelity of dendritic spine analysis and other indispensable techniques for studying neurodevelopment and plasticity. We hypothesized that EGFP’s low solubility in mammalian systems must limit the total fluorescence output of whole cells, and that improving folding efficiency could therefore translate into greater brightness of expressing neurons. By introducing rationally selected combinations of folding-enhancing mutations into GFP templates and screening for brightness and expression rate in human cells, we developed mGreenLantern, a fluorescent protein having up to sixfold greater brightness in cells than EGFP. mGreenLantern illuminates neurons in the mouse brain within 72 h, dramatically reducing lag time between viral transduction and imaging, while its high brightness improves detection of neuronal morphology using widefield, confocal, and two-photon microscopy. When virally expressed to projection neurons in vivo, mGreenLantern fluorescence developed four times faster than EYFP and highlighted long-range processes that were poorly detectable in EYFP-labeled cells. Additionally, mGreenLantern retains strong fluorescence after tissue clearing and expansion microscopy, thereby facilitating superresolution and whole-brain imaging without immunohistochemistry. mGreenLantern can directly replace EGFP/EYFP in diverse systems due to its compatibility with GFP filter sets, recognition by EGFP antibodies, and excellent performance in mouse, human, and bacterial cells. Our screening and rational engineering approach is broadly applicable and suggests that greater potential of fluorescent proteins, including biosensors, could be unlocked using a similar strategy.
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