利用果蝇模型筛选可用于动物组织光电关联的绿色荧光蛋白变体
陈 捷，石丹妮，齐佳苗，孙 玲，杨宇丰*，王道彪*
（福州大学生命科学研究所，福建 福州 350116）
摘 要 荧光蛋白能够将分子分布与超微结构联系起来，对于光电关联（CLEM）技术的应用至关重要。然而，常规的绿色荧光蛋白及其变体在活体组织的电子显微镜（EM）制样过程中，常因诸多不可控因素导致荧光信号显著减弱，这限制了它们作为光电关联分子探针的应用。本研究在果蝇大脑神经束膜胶质细胞中特异性表达多种GFP变体（mEmerald、HfYFP、mhYFP和CoGFPv0），以评估它们在活体组织光电关联中的应用潜力。通过提高表达量和靶向线粒体等策略，本研究最终筛选出适用于CLEM技术在果蝇神经系统研究中的CoGFPv0。本工作表明，CoGFPv0可作为一种具有良好应用前景的光电关联分子探针，特别适用于复杂的活体组织环境。
关键词 光电关联技术；荧光蛋白；GFP变体；CoGFPv0；果蝇
中图分类号：Q78；Q963；O657；Q336 文献标识码：BDoi:10.3969/j.issn.1000-6281.2025.01.010
Screening green fluorescent protein variants for animal tissue correlative light and electron microscopy using Drosophila melanogaster
CHEN Jie ，SHI Danni ，QI Jiamiao ，SUN Lin ，YANG Yufeng*，WANG Daobiao
（Institute of Life Sciences , Fuzhou University , Fuzhou Fujian 350116 , China）
Abstract Fluorescent proteins are crucial for correlating molecular distribution with ultrastructural details, which is essential for applications in correlative light and electron microscopy (CLEM). However, conventional green fluorescent proteins and their variants often experience significant fluorescence signal loss during electron microscopy (EM) sample preparation in live tissues, due to various uncontrollable factors, limiting their use as molecular probes for CLEM. In this study, multiple GFP variants (mEmerald, HfYFP, mhYFP, and CoGFPv0) were specifically expressed in the neuropil glial cells of the Drosophila brain to evaluate their potential for CLEM applications in vivo. Through strategies such as increasing expression levels and targeting mitochondria, this study ultimately identified CoGFPv0 as a suitable probe for CLEM in Drosophila nervous system research. This work demonstrates that CoGFPv0 shows strong potential as an effective molecular probe for CLEM, especially in complex live tissue environments.
Keywords CLEM；Fluorescent proteins；GFP variants；CoGFPv0；Drosophila
[1] GIEPMANS B N, DEERINCK T J, SMARR B L, et al. Correlated light and electron microscopic imaging of multiple endogenous proteins using quantum dots [J]. Nature Methods, 2005, 2(10): 743-749.
[2] MIRONOV A A, BEZNOUSSENKO G V. Correlative microscopy [J]. Methods in Cell Biology, 2013, 113: 209-255.
[3] JAHN K A, BARTON D A, KOBAYASHI K, et al. Correlative microscopy: Providing new understanding in the biomedical and plant sciences [J]. Micron, 2012, 43(5): 565-582.
[4] GUDMUNDSSON S, KAHLHOFER J, BAYLAC N, et al. Correlative light and electron microscopy of autophagosomes [J]. Methods in Molecular Biology, 2019, 1880: 199-209.
[5] VAN DRIEL L F, VALENTIJN J A, VALENTIJN K M, et al. Tools for correlative Cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells [J]. European Journal of Cell Biology, 2009, 88(11): 669-684.
[6] PATLA I, VOLBERG T, ELAD N, et al. Dissecting the molecular architecture of integrin adhesion sites by Cryo-electron tomography [J]. Nature Cell Biology, 2010, 12(9): 909-915.
[7] HOFFMAN D P, SHTENGEL G, XU C S, et al. Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells [J]. Science, 2020, 367(6475): eaaz5357.
[8] 陈茜，李少杰，丛铁，等. 扫描电镜用于防样品氧化样品台的设计及制备[J]. 电子显微学报，2023，42(3):352-357.
[9] 赵学平，侯小虎，刘飞，等. 扫描电镜中透射-反射式STEM明场成像装置的研制及应用[J]. 电子显微学报，2024，43(2):208-214.
[10] OORSCHOT V, LINDSEY B W, KASLIN J, et al. TEM, SEM, and STEM-based immuno-CLEM workflows offer complementary advantages [J]. Scientific Reports, 2021, 11(1): 899.
[11] OGOH K, KINEBUCHI T, MURAI M, et al. Dual-color-emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy [J]. Luminescence, 2013, 28(4): 582-591.
[12] TANIDA I, FURUTA Y, YAMAGUCHI J, et al. Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins [J]. Scientific Reports, 2020, 10(1): 21871.
[13] TSIEN R Y. The green fluorescent protein [J]. Annual Review of Biochemistry, 1998, 67: 509-544.
[14] CAMPBELL B C, PAEZ-SEGALA M G, LOOGER L L, et al. Chemically stable fluorescent proteins for advanced microscopy [J]. Nature Methods, 2022, 19(12): 1612-1621.
[15] HUANG M T, GORMAN C M. Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA [J]. Nucleic Acids Research, 1990, 18(4): 937-947.
[16] DUNCKER B P, DAVIES P L, WALKER V K. Introns boost transgene expression in Drosophila melanogaster [J]. Molecular and General Genetics, 1997, 254(3): 291-296.
[17] ZIELER H, HUYNH C Q. Intron-dependent stimulation of marker gene expression in cultured insect cells [J]. Insect Molecular Biology, 2002, 11(1): 87-95.
[18] HINK M A, GRIEP R A, BORST J W, et al. Structural dynamics of green fluorescent protein alone and fused with a single-chain Fv protein [J]. The Journal of Biological Chemistry, 2000, 275(23): 17556-17560.
