Role of CARMIL proteins in cell structure and function

CARMIL 蛋白在细胞结构和功能中的作用

• 批准号: 8746678
• 负责人: JOHN A HAMMER
• 金额: $ 62.81万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746678
• 关键词: Actins; Affinity; Animals; Ankyrin Repeat; Attenuated; Biochemical; Biological Assay; Buffers; Cell Line; Cell membrane; Cell physiology; Cells; Cellular Structures; Complex; Cytoplasm; Data; Dictyostelium; Dose; Exhibits; F-Actin; Feeds; Filament; Filopodia; Fingers; Gene Targeting; Goals; Growth; Image; In Vitro; Length; Life; Location; Microfilaments; Mus; Play; Plus End of the Actin Filament; Point Mutation; Property; Protein Binding; Proteins; RNA Cap-Binding Proteins; Reaction; Recruitment Activity; Relative (related person); Reporter; Role; Solutions; Structure; Surface; Time; Work; cellular imaging; genetic regulatory protein; in vivo; melanocyte; mutant; myotrophin; polymerization; protein function; protein purification; structural biology

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.

加帽蛋白 (CP) 通过充当肌动蛋白丝带刺末端 (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 的亲和力以及解开 CP 加帽的细丝来拮抗 CP 功能，而 V-1（肌营养蛋白）则将 CP 隔离在非活性复合物中。 先前的工作表明，CARMIL 可以轻松地从 CP: V-1 复合物中回收 CP，从而将无活性的 CP 转化为对 BE 具有中等亲和力的版本。 在这里，我们进一步阐明了这种交换反应的机制，并使用溶液测定和单丝成像证明了其对体外 BE 组装的影响。 我们表明，由复合物交换产生的 CP: CARMIL 复合物以剂量依赖的方式减慢了 BE 组装的速率，可能是通过与 BE 快速结合和解离来实现的。 此外，CP、V-1 和 CARMIL 的细胞浓度应允许它们在体内在强大的 CP 调节循环中协作。 最后，我们提供证据表明 CARMIL 被招募到质膜，并且仅在经历主动突出的细胞边缘。 假设 CARMIL 仅在该位置活跃，我们的数据表明，大量非活性 CP (CP: V-1) 通过 CARMIL 驱动的复合物交换，在血浆中形成弱封端复合物 (CP: CARMIL)突出边缘的膜。 相对于不受调节的 CP，这种机制应该增强质膜：细胞质界面处新成核的肌动蛋白丝的生长，同时保持对细胞其他地方肌动蛋白组装的强烈抑制。 Myotropin/V-1 是一种普遍表达的 13 kDa 锚蛋白重复蛋白，它以 20 nM 的亲和力以 1:1 的比例结合加帽蛋白 (CP)，形成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盘基网柄菌 (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（1 M 和 8 M） ， 分别）。 与小鼠 V-1 的生化研究一致，内源性 Dd CP 被 GST 标记的野生型 Dd V-1 (WT Dd V-1) 拉低，并由带 Flag 标记的 WT Dd V-1 共免疫沉淀。 此外，当使用包含四个紧密间隔的点突变的 Dd V-1 (FBM Dd V-1) 版本时，这些相互作用被消除，这些突变在小鼠 V-1 中大大减弱了其与 CP 的相互作用。 与 V-1 灭活 CP（肌动蛋白组装的主要细胞终止子）的能力一致，WT Dd V-1 的过度表达导致细胞总 F-肌动蛋白含量显着升高。 此外，这种增加与过度表达的程度成正比。 正如预期的那样，FBM Dd V-1 的过度表达不会改变细胞 F-肌动蛋白水平。 WT Dd V-1（但不是 FBM Dd V-1）的过度表达也会诱导富含肌动蛋白的丝状伪足结构的形成，并且这种效应再次与过度表达的程度呈正相关。 WT-DdV1过度表达不仅导致丝状伪足数量显着增加，而且还导致丝状伪足长度显着增加。此外，共同表达 F-肌动蛋白活细胞报告基因的细胞的延时图像显示，WT-DdV1 过表达细胞中的丝状伪足比对照细胞中的丝状伪足更具动态性。总之，这些过度表达研究表明，V-1 通过缓冲活性 CP 的水平来调节体内肌动蛋白聚合和丝状伪足形成（其中，在 V-1 过度表达的情况下，肌动蛋白聚合和丝状伪足形成增强，因为更多的细胞 CP中被隔离）。 这些结果与之前的研究一致，即 CP 敲低导致 B16F1 黑素细胞和盘基网柄菌中丝状伪足的爆炸性形成，并且 V-1 过度表达增强肌动蛋白聚合并诱导 PC12D 细胞中的指状表面结构。 努力创建缺乏 V-1 的盘基网柄菌细胞系，这应该进一步证实蛋白质在体内调节 CP 中的作用（这些 KO 细胞预计会表现出细胞 F-肌动蛋白含量的显着降低，因为大量细胞 CP现在将被激活）正在进行中。