Role of CARMIL proteins in cell structure and function

加帽蛋白（CP）通过作为肌动蛋白丝倒刺末端（BE）组装的主要终止子来调节肌动蛋白动力学。 CARMIL通过降低CP对BE的亲和力和通过使CP-加帽的细丝脱帽来拮抗CP功能，而V-1（肌营养因子）将CP隔离在非活性复合物中。 先前的工作表明，CARMIL可以很容易地从CP：V-1复合物中回收CP，从而将无活性的CP转化为对BE具有中等亲和力的形式。 在这里，我们进一步阐明了这种交换反应的机制，并证明了其对BE组装在体外使用溶液测定和单丝成像的效果。 我们表明，CP：通过络合物交换产生的CARMIL络合物以剂量依赖性方式减慢BE组装的速率，可能是通过快速与BE缔合和解离。 此外，CP、V-1和CARMIL的细胞浓度应允许它们在体内稳健的CP调节循环中协作。 最后，我们提供了CARMIL被募集到质膜的证据，并且仅在细胞边缘进行主动突起。 假设CARMIL仅在该位置活跃，我们的数据表明，大量的非活性CP（CP：V-1）通过CARMIL驱动的复合物交换，在质膜的突出边缘形成弱的加帽复合物（CP：CARMIL）。 这种机制应增强，相对于不受管制的CP，新的有核肌动蛋白丝在质膜：细胞质界面的生长，同时保持在细胞中的其他地方的肌动蛋白组装的强烈抑制。 肌营养蛋白/V-1是一种普遍表达的13 kDa锚定重复序列蛋白，以20 nM的亲和力1：1结合加帽蛋白（CP），产生对倒刺末端没有亲和力的复合物。 V-1的CP螯合活性可能在缓冲网骨藻（Dd）中CP的倒刺封端活性中起主要作用，特别是考虑到我们对Dd中CP和V-1的细胞浓度的估计（分别为1 M和8 M）。 与小鼠V-1的生物化学研究一致，内源性Dd CP被GST标记的野生型Dd V-1（WT Dd V-1）拉下，并被Flag标记的WT Dd V-1共免疫沉淀。 此外，当使用含有四个紧密间隔的点突变的Dd V-1（FBM Dd V-1）的版本时，这些相互作用被废除，所述点突变在小鼠V-1中极大地减弱了其与CP的相互作用。 与V-1对肌动蛋白组装的主要细胞终止子-F-CP的抑制能力一致，WT Dd V-1的过表达导致总细胞F-肌动蛋白含量的显著升高。 此外，这种增加与过度表达的程度呈正相关。 正如预期的那样，FBM Dd V-1的过表达不改变细胞F-肌动蛋白水平。 WT Dd V-1（而非FBM Dd V-1）的过度表达也诱导了富含肌动蛋白的丝状伪足样结构的形成，并且这种效应再次与过度表达的程度成正比。WT-DdV 1的过表达不仅导致丝状伪足数量的显著增加，而且导致其长度的显著增加。此外，共表达F-actin活细胞报告基因的细胞的延时图像显示，WT-DdV 1过表达细胞中的丝状伪足比对照细胞中的丝状伪足更动态。总之，这些过表达研究表明，V-1调节肌动蛋白聚合和丝状伪足形成在体内通过缓冲活性CP的水平（其中，在V-1过表达的情况下，肌动蛋白聚合和丝状伪足形成增强，因为更多的细胞CP被隔离）。 这些结果与先前的研究一致，表明CP敲低导致B16 F1黑素细胞和Dictyosteoprotein中丝状伪足的爆炸性形成，并且V-1过表达增强肌动蛋白聚合并诱导PC 12 D细胞中的指状表面结构。 正在努力创建缺乏V-1的网骨藻细胞系，这将进一步证实蛋白质在体内调节CP中的作用（这些KO细胞预计表现出细胞F-肌动蛋白含量的显著下降，因为大部分细胞CP现在将是活性的）。

Capping Protein (CP) regulates actin dynamics by serving as the major terminator of assembly at the actin filament barbed end (BE). CARMIL antagonizes CP function by reducing CPs affinity for the BE 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 BE. Here we further clarify the mechanism of this exchange reaction, and demonstrate its effect on BE assembly in vitro using solution assays and single filament imaging. We show that the CP: CARMIL complex created by complex exchange slows in a dose-dependent manner the rate of BE assembly, probably by rapidly associating with, and dissociating from, the BE. Moreover, the cellular concentrations of CP, V-1 and CARMIL should allow them to collaborate in a robust CP regulatory cycle in vivo. Finally, we provide evidence 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 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. This mechanism should enhance, relative to unregulated CP, the growth of newly-nucleated actin filaments at the plasma membrane: cytoplasm interface, while maintaining strong suppression of actin assembly elsewhere in the cell. Myotrophin/V-1 is a ubiquitously expressed, 13 kDa ankyrin-repeat protein that binds Capping Protein (CP) 1:1 with an affinity of 20 nM, resulting in a complex that has no affinity for the barbed end. The CP sequestering activity of V-1 may play a major role in buffering the barbed end capping activity of CP in Dictyostelium (Dd), especially given our estimates of the cellular concentrations of CP and V-1 in Dd (1 M and 8 M, respectively). Consistent with biochemical studies of mouse V-1, endogenous Dd CP is pulled down by GST-tagged, wild type Dd V-1 (WT Dd V-1) and is co immuno- precipitated by Flag-tagged, WT Dd V-1. Moreover, these interactions are abrogated when using a version of Dd V-1 (FBM Dd V-1) containing four closely-spaced point mutations that in mouse V-1 greatly attenuate its interaction with CP. Consistent with V-1s ability to inactivate CP, the major cellular terminator of actin assembly, the over expression of WT Dd V-1 results in a significant elevation in total cellular F-actin content. Moreover, this increase scales positively with degree of over expression. As expected, the over expression of FBM Dd V-1 does not alter cellular F-actin levels. The over expression of WT Dd V-1 (but not FBM Dd V-1) also induces the formation of actin-rich, filopodial-like structures, and this effect once again scales positively with the degree of over expression. WT-DdV1 over expression leads not only to a significant increase in the number of filopodia, but also to a significant increase in their length. Moreover, time lapse images of cells co expressing a live-cell reporter for F-actin reveal that the filopodia in WT-DdV1 over-expressing cells are more dynamic than in control cells. Together, these over expression studies suggest that V-1 regulates actin polymerization and filopdial formation in vivo by buffering the level of active CP (where, in the case of V-1 over expression, actin polymerization and filopodia formation are enhanced because more cellular CP in sequestered). These results are consistent with previous studies showing that CP knockdown leads to the explosive formation of filopodia in both B16F1 melanocytes and Dictyostelium, and that V-1 over expression enhances actin polymerization and induces finger-like surface structures in PC12D cells. Efforts to create a Dictyostelium cell line that lacks V-1, which should provide further confirmation of the proteins role in regulating CP in vivo (these KO cells are expected to exhibit a profound decease in cellular F-actin content because the bulk of cellular CP will now be active) are underway.