Department of Biology, Reed College, Portland, OR 97202.
Department of Biology & Integrative Program for Biological and Genome Sciences, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280.
Mol Biol Cell. 2020 Oct 1;31(21):2379-2397. doi: 10.1091/mbc.E20-03-0181. Epub 2020 Aug 20.
To identify novel regulators of nonmuscle myosin II (NMII) we performed an image-based RNA interference screen using stable S2 cells expressing the enhanced green fluorescent protein (EGFP)-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. We identified the Rab-specific GTPase-activating protein (GAP) RN-tre as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1 and a decrease in phosphomyosin-positive cells by immunostaining, while expression of constitutively active Rho or Rho-kinase (Rok) rescues the punctate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as overexpression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, our results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility.
为了鉴定非肌肉肌球蛋白 II（NMII）的新调节因子，我们利用稳定表达增强型绿色荧光蛋白（EGFP）标记的 NMII 调节轻链（RLC）和 mCherry-Actin 的 S2 细胞进行了基于图像的 RNA 干扰筛选。我们发现 Rab 特异性 GTPase 激活蛋白（GAP）RN-tre 对于 NMII RLC 组装到收缩性肌动蛋白网络中是必需的。RN-tre 的耗竭导致 NMII 出现点状表型，类似于 Rho1 途径蛋白耗竭后观察到的表型。RN-tre 的耗竭还导致活性 Rho1 减少和免疫染色的磷酸化肌球蛋白阳性细胞减少，而组成性激活的 Rho 或 Rho-kinase（Rok）的表达可挽救点状表型。功能上，RN-tre 的耗竭分别导致 S2 和 S2R+细胞中肌动蛋白逆行流动速率和细胞收缩性增加。RN-tre 对 NMII 的调节仅部分依赖于其 GAP 活性，因为 RN-tre 失活的组成性激活 Rab 过表达未能改变 NMII RLC 定位，而 RN-tre 的 GAP 失活版本部分恢复了磷酸化肌球蛋白染色。总之，我们的结果表明，RN-tre 在 NMII RLC 分布、磷酸化和功能中发挥重要的调节作用，可能通过 Rho1 信号转导，并可能作为分泌机制和肌球蛋白收缩性之间的联系。
