Smith Samantha L, Iwamoto Yuichiro, Manimaran Aadhithya, Drubin David G
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
bioRxiv. 2025 Jun 27:2025.06.25.661608. doi: 10.1101/2025.06.25.661608.
Genome editing has enabled the integration of fluorescent protein coding sequences into genomes, resulting in expression of in-frame fusion proteins under the control of their natural gene regulatory sequences. While this technique overcomes the well-documented artifacts associated with gene overexpression, editing genomes of metazoan cells incurs a significant time cost compared to simpler organisms, such as yeast. Editing two or more genes to express multiple fluorescent fusion proteins in a single cell line has proven to be a powerful strategy for uncovering spatio-dynamic, and therefore functional, relationships among different proteins, but it can take many months to edit each gene within the same cell line. Here, by utilizing cell fusions, we quickly generated cells expressing pairwise permutations of fluorescent fusion proteins in genome-edited human cells to reveal previously undetected protein-organelle interactions. We fused human induced pluripotent stem cells (hiPSCs) that express in-frame fusions of clathrin-mediated endocytosis (CME) and actin cytoskeleton proteins with hiPSCs that express fluorescently tagged organelle markers, uncovering novel interactions between CME proteins, branched actin filament networks, and lysosomes.
基因组编辑技术已能够将荧光蛋白编码序列整合到基因组中，从而在其天然基因调控序列的控制下表达读码框内融合蛋白。虽然这项技术克服了与基因过表达相关的诸多人为干扰因素，但与酵母等较为简单的生物体相比，编辑后生动物细胞的基因组会耗费大量时间。在单一细胞系中编辑两个或更多基因以表达多种荧光融合蛋白，已被证明是揭示不同蛋白质之间时空动态关系（进而揭示功能关系）的有力策略，但在同一细胞系中编辑每个基因可能需要数月时间。在此，我们利用细胞融合技术，在经过基因组编辑的人类细胞中快速生成了表达荧光融合蛋白两两排列组合的细胞，以揭示此前未被发现的蛋白质与细胞器之间的相互作用。我们将表达网格蛋白介导的内吞作用（CME）和肌动蛋白细胞骨架蛋白读码框内融合蛋白的人类诱导多能干细胞（hiPSC）与表达荧光标记细胞器标志物的hiPSC进行融合，从而发现了CME蛋白、分支肌动蛋白丝网络和溶酶体之间的新相互作用。
