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.

封闭蛋白（CP）通过作为肌动蛋白丝末端（BE）的主要终结剂（BE）来调节肌动蛋白动力学。 Carmil通过降低对BE的CPS亲和力和解染CP覆盖的细丝来拮抗CP功能，而V-1（肌营养素）隔离CP在不活跃的复合物中隔离CP。 先前的工作表明，卡米尔可以轻松从CP：V-1复合物中检索CP，从而将非活性CP转换为对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细丝成像证明了其对体外组装的影响。 我们表明，由复杂交换产生的CP：Carmil复合物以剂量依赖性的方式减慢了组装的速率，可能是通过与BE迅速相关并分离的。 此外，CP，V-1和Carmil的细胞浓度应允许它们在体内稳健的CP调节周期中进行协作。 最后，我们提供了证据表明卡米尔被募集到质膜，并且仅在经历活跃突出的细胞边缘。 假设Carmil仅在此位置处于活动状态，我们的数据认为，通过Carmil驱动的复合物交换，大量的无活性CP（CP：V-1）饲料是在突出边缘的质膜上形成弱封闭复合物（CP：Carmil）的形成。 相对于不受管制的CP，这种机制应增强质膜上新近核肌动蛋白丝的生长：细胞质界面，同时保持细胞其他地方其他地方的肌动蛋白组件的强抑制作用。 肌营养蛋白/V-1是一种普遍表达的，13 kDa arnkyrin-repeat蛋白，结合了封盖蛋白（CP）1：1，亲和力为20 nm，导致一个对带状端端具有亲和力的复杂性。 V-1的CP隔离活性在缓冲DICTYOSTELIUM（DD）的刺端末端封盖活性方面可能起主要作用，尤其是考虑到我们对DD中CP和V-1的细胞浓度的估计值（分别为1 m和8 m）。 与小鼠V-1的生化研究一致，内源性DD CP通过GST标记的野生型DD V-1（WT DD V-1）拉下，并通过Flag标记的WT DD V-1降压。 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）的版本时，这些相互作用将被废除，这些突变大大减弱了其与CP的相互作用。 与V-1灭活CP（肌动蛋白组装的主要细胞终结剂）的能力一致，WT DD DD V-1的过度表达会导致总细胞F-肌动蛋白含量显着升高。 此外，这种增加的尺度会随着过度表达程度而积极。 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）的过度表达也诱导了富含肌动蛋白，丝状菌样结构的形成，并且这种效应再次随着过度表达的程度呈阳性。 WT-DDV1超过表达不仅会导致丝状虫的数量显着增加，而且导致其长度显着增加。此外，表达F-肌动蛋白的活细胞报告基因的细胞的延时图像表明，WT-DDV1过表达的细胞中的丝状细胞比对照细胞中更动态。总之，这些过度表达的研究表明，V-1通过缓冲活性CP的水平（在V-1的情况下，在表达过高，肌动蛋白聚合和丝状卵形形成的情况下，可以增强体内肌动蛋白聚合和丝状形成，因为在序列中更细胞的CP）。 这些结果与先前的研究相一致，表明CP敲低导致B16F1黑色素细胞和DICSYOSTELIUM的丝状爆炸形成，并且V-1过度表达增强了肌动蛋白的聚合化并诱导PC12D细胞中的手指样表面结构。 创建缺乏V-1的Dictyostelium细胞系的努力，该细胞系应进一步确认蛋白质在调节体内CP中的作用（这些KO细胞有望在细胞F-肌动蛋白含量中表现出深刻的脱发，因为大部分细胞CP现在将活跃）。