Keratin-binding ability of the N-terminal Solo domain of Solo is critical for its function in cellular mechanotransduction
Keratin-binding ability of the N-terminal Solo domain of Solo is critical for its function in cellular mechanotransduction
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DOI：
10.1111/gtc.12682
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发表时间：
2019-05-01
期刊：
影响因子：
2.1
通讯作者：
Deguchi, Shinji
中科院分区：
文献类型：
--
作者：
Fujiwara, Sachiko;Matsui, Tsubasa S.;Deguchi, Shinji
Solo（ARHGEF 40）是一种RhoA靶向鸟嘌呤核苷酸交换因子，可调节张力诱导的细胞骨架重组。Solo通过多个位点与角蛋白8/角蛋白18（K8/K18）细丝结合，但这些相互作用在Solo的定位和机械转导调节功能中的作用仍不清楚。在这里，我们构建了两个Solo突变体（L14 R/L17 R和L49 R/L52 R），在N-末端保守区（我们称之为Solo结构域）中具有亮氨酸到精氨酸的替换，并分析了它们的K18结合活性。这些突变显著降低了Solo N-末端片段（残基1-329）的K18结合能力，但对全长（FL）Solo的K18结合能力没有明显影响。当在培养的细胞中表达时，野生型Solo-FL在细胞的腹侧表面附近显示出独特的点状定位，并引起肌动蛋白丝的增强。相反，尽管保留了K18结合能力，L14 R/L17 R和L49 R/L52 R突变体的Solo-FL弥漫分布在细胞质中，几乎没有诱导肌动蛋白细胞骨架的加强。此外，野生型Solo-FL促进了对细胞外基质的牵引力产生和张力诱导的应力纤维增强，但其L14 R/L17 R和L49 R/L52 R突变体没有。这些结果表明，K18的N-末端Solo结构域的结合能力是至关重要的腹侧定位的Solo和其功能，在调节mechanotransduction。
Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor that regulates tensional force-induced cytoskeletal reorganization. Solo binds to keratin 8/keratin 18 (K8/K18) filaments through multiple sites, but the roles of these interactions in the localization and mechanotransduction-regulating function of Solo remain unclear. Here, we constructed two Solo mutants (L14R/L17R and L49R/L52R) with leucine-to-arginine replacements in the N-terminal conserved region (which we termed the Solo domain) and analyzed their K18-binding activities. These mutations markedly decreased the K18-binding ability of the N-terminal fragment (residues 1-329) of Solo but had no apparent effect on the K18-binding ability of full-length (FL) Solo. When expressed in cultured cells, wild-type Solo-FL showed a unique punctate localization near the ventral surface of cells and caused the reinforcement of actin filaments. In contrast, despite retaining the K18-binding ability, the L14R/L17R and L49R/L52R mutants of Solo-FL were diffusely distributed in the cytoplasm and barely induced actin cytoskeletal reinforcement. Furthermore, wild-type Solo-FL promoted traction force generation against extracellular matrices and tensional force-induced stress fiber reinforcement, but its L14R/L17R and L49R/L52R mutants did not. These results suggest that the K18-binding ability of the N-terminal Solo domain is critical for the ventral localization of Solo and its function in regulating mechanotransduction.