Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
Department of Cell Biology and Physiology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
PLoS One. 2019 Sep 4;14(9):e0221962. doi: 10.1371/journal.pone.0221962. eCollection 2019.
Vinculin (Vcn) is a ubiquitously expressed cytoskeletal protein that links transmembrane receptors to actin filaments, and plays a key role in regulating cell adhesion, motility, and force transmission. Metavinculin (MVcn) is a Vcn splice isoform that contains an additional exon encoding a 68-residue insert within the actin binding tail domain. MVcn is selectively expressed at sub-stoichiometic amounts relative to Vcn in smooth and cardiac muscle cells. Mutations in the MVcn insert are linked to various cardiomyopathies. In vitro analysis has previously shown that while both proteins can engage filamentous (F)-actin, only Vcn can promote F-actin bundling. Moreover, we and others have shown that MVcn can negatively regulate Vcn-mediated F-actin bundling in vitro. To investigate functional differences between MVcn and Vcn, we stably expressed either Vcn or MVcn in Vcn-null mouse embryonic fibroblasts. While both MVcn and Vcn were observed at FAs, MVcn-expressing cells had larger but fewer focal adhesions per cell compared to Vcn-expressing cells. MVcn-expressing cells migrated faster and exhibited greater persistence compared to Vcn-expressing cells, even though Vcn-containing FAs assembled and disassembled faster. Magnetic tweezer measurements on Vcn-expressing cells show a typical cell stiffening phenotype in response to externally applied force; however, this was absent in Vcn-null and MVcn-expressing cells. Our findings that MVcn expression leads to larger but fewer FAs per cell, in conjunction with the inability of MVcn to bundle F-actin in vitro and rescue the cell stiffening response, are consistent with our previous findings of actin bundling deficient Vcn variants, suggesting that deficient actin-bundling may account for some of the differences between Vcn and MVcn.
波形蛋白（Vinculin，Vcn）是一种广泛表达的细胞骨架蛋白，它将跨膜受体与肌动蛋白丝连接起来，在调节细胞黏附、运动和力传递方面发挥着关键作用。Metavinculin（MVcn）是 Vcn 的剪接异构体，其中包含一个额外的外显子，在肌动蛋白结合尾部结构域中编码一个 68 个残基的插入片段。MVcn 在平滑肌和心肌细胞中以低于 Vcn 的亚化学计量表达。MVcn 插入片段的突变与各种心肌病有关。体外分析先前表明，尽管这两种蛋白都可以与丝状肌动蛋白（F-actin）结合，但只有 Vcn 可以促进 F-actin 成束。此外，我们和其他人已经表明，MVcn 可以在体外负调控 Vcn 介导的 F-actin 成束。为了研究 MVcn 和 Vcn 之间的功能差异，我们在 Vcn 缺失的小鼠胚胎成纤维细胞中稳定表达 Vcn 或 MVcn。尽管在 FA 中都观察到了 MVcn 和 Vcn，但与表达 Vcn 的细胞相比，表达 MVcn 的细胞每细胞的 FA 更大但数量更少。与表达 Vcn 的细胞相比，表达 MVcn 的细胞迁移更快且具有更大的持续性，尽管含 Vcn 的 FA 组装和拆卸更快。在表达 Vcn 的细胞上进行的磁镊测量显示出对外力的典型细胞变硬表型；然而，这种现象在 Vcn 缺失和 MVcn 表达的细胞中不存在。我们的发现表明，MVcn 的表达导致每细胞的 FA 更大但数量更少，并且 MVcn 在体外不能成束 F-actin 并且不能挽救细胞变硬反应，这与我们先前发现的肌动蛋白成束缺陷的 Vcn 变体一致，表明肌动蛋白成束缺陷可能是 Vcn 和 MVcn 之间的一些差异的原因。
