Neutrophil-like HL-60 cells expressing only GFP-tagged β-actin exhibit nearly normal motility
Neutrophil-like HL-60 cells expressing only GFP-tagged β-actin exhibit nearly normal motility
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DOI：
10.1002/cm.21603
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发表时间：
2020-02-28
期刊：
影响因子：
2.9
通讯作者：
Theriot, Julie A.
中科院分区：
文献类型：
--
作者：
Garner, Rikki M.;Skariah, Gemini;Theriot, Julie A.
使用荧光显微镜观察活细胞中的肌动蛋白动力学是探索细胞迁移机制的基础。我们使用 CRISPR/Cas9 基因编辑生成内源基因座 (ACTB) 表达 GFP-β-肌动蛋白的中性粒细胞样 HL-60 细胞系。鉴于许多先前概述标记肌动蛋白功能缺陷的报告，我们预计 HL-60 细胞只能耐受单等位基因编辑，因为双等位基因编辑细胞不会产生正常的 β-肌动蛋白。令人惊讶的是，我们恢复了活的单等位基因 GFP-β-肌动蛋白细胞以及双等位基因编辑的 GFP-β-肌动蛋白细胞，其中一个 ACTB 基因拷贝被沉默，另一个包含 GFP 标签。此外，在各种运动测定中，编辑的细胞以与未修饰的细胞相似的速度和持久性迁移，并且具有几乎正常的细胞形状。这些结果可能部分地可以通过我们的观察来解释，即 GFP-β-肌动蛋白在双等位基因编辑的细胞中以与未编辑细胞中的正常 β-肌动蛋白相似的效率并入 F-肌动蛋白网络。此外，编辑后的细胞显着上调γ-肌动蛋白，可能有助于补偿正常β-肌动蛋白的损失。有趣的是，根据 RNA 测序的测量，相对于单等位基因系，双等位基因编辑的细胞在整体基因表达方面仅发生了适度的变化。虽然单等位基因编辑的细胞下调标记等位基因的表达，因此仅发出微弱的荧光，但双等位基因编辑的细胞非常明亮，非常适合活细胞显微镜检查。这种荧光标记方法的非破坏性表型和直接可解释性使其成为研究这些快速迁移和高度吞噬细胞中肌动蛋白动力学的有前途的工具。
Observations of actin dynamics in living cells using fluorescence microscopy have been foundational in the exploration of the mechanisms underlying cell migration. We used CRISPR/Cas9 gene editing to generate neutrophil-like HL-60 cell lines expressing GFP-beta-actin from the endogenous locus (ACTB). In light of many previous reports outlining functional deficiencies of labeled actin, we anticipated that HL-60 cells would only tolerate a monoallelic edit, as biallelic edited cells would produce no normal beta-actin. Surprisingly, we recovered viable monoallelic GFP-beta-actin cells as well as biallelic edited GFP-beta-actin cells, in which one copy of the ACTB gene is silenced and the other contains the GFP tag. Furthermore, the edited cells migrate with similar speeds and persistence as unmodified cells in a variety of motility assays, and have nearly normal cell shapes. These results might partially be explained by our observation that GFP-beta-actin incorporates into the F-actin network in biallelic edited cells at similar efficiencies as normal beta-actin in unedited cells. Additionally, the edited cells significantly upregulate gamma-actin, perhaps helping to compensate for the loss of normal beta-actin. Interestingly, biallelic edited cells have only modest changes in global gene expression relative to the monoallelic line, as measured by RNA sequencing. While monoallelic edited cells downregulate expression of the tagged allele and are thus only weakly fluorescent, biallelic edited cells are quite bright and well-suited for live cell microscopy. The nondisruptive phenotype and direct interpretability of this fluorescent tagging approach make it a promising tool for studying actin dynamics in these rapidly migrating and highly phagocytic cells.