Role of CARMIL proteins in cell structure and function

哺乳动物肌营养蛋白/V-1是一种13 kDa的锚蛋白重复蛋白，在体外以20 nM的亲和力与Capping蛋白（CP）结合，形成1:1复合物（CP: V-1），对倒刺端没有亲和力。因此，V-1有可能通过减少倒钩端盖的程度来影响肌动蛋白在体内的聚合。盘状盘基骨柱（Dictyostelium disideum, d.d.）含有一个编码13.2 kDa蛋白的单基因，与小鼠V-1具有高度的序列相似性。与小鼠V-1一样，Dd V-1与CP紧密结合，在细胞中以比CP多三倍的摩尔量存在，当将功能阻断点突变（FBM）引入第一个锚蛋白环（FBM Dd V-1）时，Dd V-1不再与CP结合。与V-1隔离CP的能力一致，Dd V-1的过表达导致细胞f -肌动蛋白总含量升高，并与过表达程度成正比。重要的是，FBM Dd V-1的过表达不会改变细胞f -肌动蛋白水平，这表明V-1过表达的影响是由于其隔离CP的能力。Dd V-1的过表达，而不是FBM-Dd V-1，也会诱导肌动蛋白丰富的丝状样结构的形成，这种结构与过表达程度成正比。与过表达相比，同源重组产生的Dd V-1零细胞显示细胞f -肌动蛋白含量大幅下降。此外，这些细胞的生长速度、巨噬细胞率、趋化流效率、迁移过程中的极性和随机运动率显著降低。重要的是，这些缺陷是由野生型V-1拯救的，而不是由FBM Dd V-1。总之，这些结果表明，V-1在调节细胞中肌动蛋白的组装中起主要作用。此外，FBM V-1既没有诱导过表达表型，也没有挽救空细胞表型，这表明V-1通过缓冲细胞CP来影响肌动蛋白细胞骨架。因此，我们得出结论，V-1的过表达和敲除分别导致细胞CP活性的降低和升高，从而导致细胞肌动蛋白含量的变化，这与CPs倒钩端盖住活性一致。此外，这些变化的功能后果，包括依赖于肌动蛋白组装的许多细胞活动的改变，表明V-1在生理上是重要的。

Mammalian myotrophin/V-1 is a 13 kDa, ankyrin-repeat protein that binds Capping Protein (CP) in vitro with an affinity of 20 nM, creating a 1:1 complex (CP: V-1) that has no affinity for the barbed end. V-1 has the potential, therefore, to influence actin polymerization in vivo by reducing the extent of barbed end capping. Dictyostelium discoideum (D.d.) contains a single gene encoding a 13.2 kDa protein with high sequence similarity to mouse V-1. Like mouse V-1, Dd V-1 binds CP tightly, is present in cells at a three-fold molar excess over CP, and no longer binds CP when a function-blocking point mutation (FBM) is introduced into the first ankyrin loop (FBM Dd V-1). Consistent with V-1s ability to sequester CP, over-expression of Dd V-1 results in an elevation in total cellular F-actin content that scales positively with the degree of over-expression. Importantly, over-expression of FBM Dd V-1 does not alter cellular F-actin levels, arguing that the effect of V-1 over-expression is due to its ability to sequester CP. The over-expression of Dd V-1, but not FBM-Dd V-1, also induces the formation of actin-rich, filopodial-like structures that scales positively with the degree of over-expression. In contrast to over-expression, Dd V-1 null cells created by homologous recombination exhibit a large decrease in cellular F-actin content. Moreover, these cells exhibit significant decreases in growth rate, macropinocytosis rate, chemotactic streaming efficiency, polarity during migration, and random motility rate. Importantly, these defects are rescued by wild type V-1 but not by FBM Dd V-1. Together, these results argue that V-1 plays a major role in regulating actin assembly in cells. Moreover, the fact that FBM V-1 neither induces over expression phenotypes nor rescues null cell phenotypes argues that V-1 exerts its effects on the actin cytoskeleton by buffering cellular CP. We conclude, therefore, that reductions and elevations in cellular CP activity caused by the over-expression and knock out of V-1, respectively, result in changes in cellular actin content that are consistent with CPs barbed end capping activity. Moreover, the functional consequences of these changes, which involve alterations in numerous cellular activities that dependent on actin assembly, indicate that V-1 is physiologically important. While Capping Protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, CARMIL antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, while V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP: V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP: CARMIL complex created by complex exchange slows the rate of barbed end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane, and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely-diffusing, inactive CP (CP: V-1) feeds, via CARMIL-driven complex exchange, the formation of weak capping complexes (CP: CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed end capping there.