Kurashina Mizuki, Snow Andrew W, Mizumoto Kota
Graduate Program of Cell and Developmental Biology, Life Sciences Institute, The University of British Columbia, Vancouver, Canada V6T 1Z3.
Department of Zoology, The University of British Columbia, Vancouver, Canada V6T 1Z3.
Genetics. 2025 Mar 17;229(3). doi: 10.1093/genetics/iyae214.
Visualizing the subcellular localization of presynaptic proteins with fluorescent proteins is a powerful tool to dissect the genetic and molecular mechanisms underlying synapse formation and patterning in live animals. Here, we utilize split green and red fluorescent proteins to visualize the localization of endogenously expressed presynaptic proteins at a single-neuron resolution in Caenorhabditis elegans. By using CRISPR/Cas9 genome editing, we generated a collection of C. elegans strains in which endogenously expressed presynaptic proteins (RAB-3/Rab3, SNG-1/Synaptogyrin, CLA-1/Piccolo, SYD-2/Liprin-α, UNC-10/RIM, RIMB-1/RIM-BP, and ELKS-1/ELKS) are tagged with tandem repeats of GFP11 and/or wrmScarlet11. We show that the expression of GFP1-10 and wrmScarlet1-10 under neuron-specific promoters can robustly label presynaptic proteins in different neuron types. We believe that the combination of our knock-in strains and GFP1-10 and wrmScarlet1-10 plasmids is a versatile modular system useful for neuroscientists to examine the localization of endogenous presynaptic proteins in any neuron type in C. elegans.
利用荧光蛋白可视化突触前蛋白的亚细胞定位是剖析活体动物中突触形成和模式化潜在遗传和分子机制的有力工具。在这里，我们利用分裂的绿色和红色荧光蛋白，以单个神经元分辨率在秀丽隐杆线虫中可视化内源性表达的突触前蛋白的定位。通过使用CRISPR/Cas9基因组编辑，我们构建了一系列秀丽隐杆线虫菌株，其中内源性表达的突触前蛋白（RAB-3/Rab3、SNG-1/突触旋转蛋白、CLA-1/ piccolo、SYD-2/脂联蛋白-α、UNC-10/RIM、RIMB-1/RIM-BP和ELKS-1/ELKS）用GFP11和/或wrmScarlet11的串联重复序列进行标记。我们表明，在神经元特异性启动子下GFP1-10和wrmScarlet1-10的表达可以强有力地标记不同神经元类型中的突触前蛋白。我们相信，我们的敲入菌株与GFP1-10和wrmScarlet1-10质粒的组合是一个通用的模块化系统，有助于神经科学家检查秀丽隐杆线虫中任何神经元类型内源性突触前蛋白的定位。
