CEA, iRTSV, Laboratoire Physiologie Cellulaire & Végétale, CNRS, UMR5168, Université Joseph Fourier-Grenoble I, F-38054 Grenoble, France.
Mol Biol Cell. 2011 Dec;22(23):4575-87. doi: 10.1091/mbc.E11-07-0616. Epub 2011 Oct 12.
A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.
许多细胞过程既利用微管又利用肌动蛋白丝，但将这两种细胞骨架元件连接起来的分子机制仍有待详细阐明。formin 是一种肌动蛋白结合蛋白，对肌动蛋白动力学有多种影响，一种formin，mDia2，已被证明通过其formin 同源结构域 2（FH2）结合并稳定微管。在这里，我们表明三种formin，INF2、mDia1 和 mDia2，在与微管和肌动蛋白的相互作用中显示出重要的差异。包含所有三种formin 的 FH1、FH2 和 C 端结构域的构建体以高亲和力（K(d) < 100 nM）结合微管。然而，只有 mDia2 以 1:1 化学计量结合微管，INF2 和 mDia1 以约 1:3 （formin 二聚体：微管二聚体）饱和结合。INF2-FH1FH2C 是一种有效的微管束蛋白，这一效应导致突发率大大降低。相比之下，mDia1 和 mDia2 都不是有效的微管束蛋白。mDia2 和 INF2 的 C 端在微管相互作用中有不同的功能，mDia2 的 C 端需要高亲和力结合，而 INF2 的 C 端需要束集。mDia2 的 C 端以亚微摩尔亲和力直接结合微管。这些formin 在同时结合肌动蛋白和微管的能力上也存在差异。微管强烈抑制 mDia2 的肌动蛋白聚合，而它们适度抑制 mDia1 并且对 INF2 没有影响。相反，肌动蛋白单体抑制 INF2 结合/束集微管，但不影响 mDia1 或 mDia2。这些与微管和肌动蛋白相互作用的差异表明，在需要这两种细胞骨架元件的细胞过程中具有不同的功能。
