Structure And Function Of Unconventional Myosins and CARMIL Proteins

非常规肌球蛋白和 CARMIL 蛋白的结构和功能

• 批准号: 7968965
• 负责人: JOHN A HAMMER
• 金额: $ 214.66万
• 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/7968965
• 关键词: ATP Hydrolysis; Acanthamoeba; Actins; Address; Affinity; Amoeba genus; Animals; Aspergillus; Automobile Driving; Biochemistry; Biological; Biological Assay; Biological Models; Bladder; Cell physiology; Cells; Complex; Cytoskeleton; Dendritic Spines; Dictyostelium; Dissociation; Endoplasmic Reticulum; Ensure; Exhibits; Filament; Genes; Goals; Image; Individual; Kinetics; Lymphocyte; MYO5A gene; Measurable; Mediating; Membrane; Microfilaments; Microtubule-Organizing Center; Microtubules; Modeling; Motor; Movement; Mus; Mutation; Myosin ATPase; Myosin Type V; Neurons; Normal Statistical Distribution; Null Lymphocytes; Organelles; Osmoregulation; Proteins; Recombinants; Regulation; Role; Signal Pathway; Solutions; Structure; Synaptic plasticity; Time; Total Internal Reflection Fluorescent; Transmembrane Transport; Vacuole; Vertebral column; Work; actin capping protein; attenuation; base; cell motility; cell type; in vivo; melanocyte; mutant; polymerization; restoration; single molecule; structural biology; time interval

Extension of the endoplasmic reticulum (ER) network into dendritic spines of Purkinje neurons (PNs) is required for cerebellar synaptic plasticity and disrupted in animals with mutations in MYO5A, the gene encoding the class V myosin Myosin Va. Notably, the mechanism ensuring the ERs localization to spines has not been unraveled. While it has been proposed that animal class V myosins localize organelles by tethering them to the actin cytoskeleton, we demonstrate here that Myosin Va is an organelle transporter that pulls ER as cargo into PN spines. We show that the myosin accumulates at the ER tip as the organelle moves into spines, and that the myosins ability to hydrolyze ATP is required for the ERs movement into spines. Importantly, we provide direct proof for Myosin Va driving ER motility, as an attenuation of the myosins capability to move along actin filaments reduces the velocity of ER movement. Thus, we establish for the first time within animal cells that an actin-based motor is moving ER, and we thereby uncover the basis of the mechanism that mediates ER localization to PN spines, a prerequisite for synaptic plasticity. The contractile vacuole (CV) complex in Dictyostelium is a tubulovesicular osmoregulatory organelle that exhibits extensive motility along the actin-rich cortex, providing a useful model for investigating myosin-dependent membrane transport. Here we show that the type V myosin myoJ localizes to CV membranes and is required for efficient osmoregulation, the normal accumulation of CV membranes in the cortex, and the conversion of collapsed bladder membranes into outwardly radiating cortical CV tubules. Complementation of myoJ null cells with mechanochemically compromised versions of myoJ results in predictable changes in the dynamics of these radiating tubules, confirming myoJs role in moving CV membranes along the cortex. MyoJ null cells also exhibit a dramatic concentration of CV membranes around the MTOC. Consistently, we demonstrate that CV membranes also move bi-directionally on microtubules between the cortex and the MTOC. Therefore, myoJ cooperates with plus and minus end-directed microtubule motors to drive the normal distribution and dynamics of the CV complex in Dictyostelium. Bulk solution assays have shown that the isolated CAH3 domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with Capping Protein (CP). To demonstrate this putative uncapping activity directly, we used TIRF microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). Addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric GFP (mGFP-CP). Restoration of polymerization upon mCAH3 addition was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of 90% at 250 nM mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from 30 minutes without mCAH3 to 10 seconds with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL, and presumably the intact molecule as well, possesses the ability to uncap CP-capped actin filaments.

内质网(ER)网络延伸到浦肯野神经元(PNS)树突棘是小脑突触可塑性所必需的，在编码V类肌球蛋白Myosin Va基因MYO5A突变的动物中，内质网(ER)网络的延伸是必需的。值得注意的是，确保内质网定位于脊柱的机制尚未解开。虽然有人提出动物V类肌球蛋白通过将细胞器拴在肌动蛋白细胞骨架上来定位细胞器，但我们在这里证明了肌球蛋白Va是一种细胞器转运体，将内质网作为货物运送到PN脊柱中。我们表明，肌球蛋白在细胞器移动到脊椎时在内质网末端积累，并且肌球蛋白水解三磷酸腺苷的能力是内质网移动到脊柱中所必需的。重要的是，我们提供了肌球蛋白Va驱动内质网运动的直接证据，因为肌球蛋白沿肌动蛋白细丝移动的能力减弱会降低内质网运动的速度。因此，我们首次在动物细胞中建立了基于肌动蛋白的马达正在移动内质网，从而揭示了将内质网定位到PN脊椎的机制的基础，这是突触可塑性的先决条件。 网茎中的收缩空泡(CV)复合体是一种沿富含肌动蛋白的皮质广泛运动的管状泡状渗透调节细胞器，为研究肌球蛋白依赖的膜转运提供了一个有用的模型。在这里，我们证明了V型肌球蛋白myoJ定位于CV膜，是有效的渗透调节、CV膜在皮质中的正常积累以及将塌陷的膀胱膜转化为向外辐射的皮质CV管所必需的。用机械力化学折衷版本的myoJ来补充myoJ零细胞，导致这些辐射小管的动力学发生可预见的变化，证实了myoJ在沿皮质移动CV膜中的作用。MyoJ零细胞还在MTOC周围显示出大量的CV膜。我们一直证明，在皮质和MTOC之间的微管上，CV膜也是双向移动的。因此，myoJ与正、负端导向的微管马达协同作用，驱动网柄网柄菌内CV复合体的正常分布和动态变化。 大量溶液分析表明，从小鼠和棘阿米巴中分离出的CAH3结构域，当加入到先前由帽蛋白(CP)覆盖的肌动蛋白细丝中时，可以快速而有效地恢复肌动蛋白的聚合。为了直接证明这一假定的去封顶活性，我们使用TIRF显微镜观察了添加小鼠CARMIL-1的CAH3结构域(MCAH3)前后的单个CP封顶的肌动蛋白细丝。MCAH3的加入迅速恢复了单个封端长丝的聚合，与未封端一致。为了验证去封顶，用标记有单体GFP的重组小鼠CP(mGFP-CP)封端细丝。在mCAH3加入后立即恢复聚合之前，mGFP-CP从丝端完全解离，证实了CAH3驱动的解帽机制。定量分析表明，随着mCAH3浓度的增加，未封端的细丝比例增加，在250 nM的mCAH3处达到最大值90%。此外，随着mCAH3浓度的增加，mCAH3添加到解帽的时间间隔缩短，CP在带刺末端的半衰期从没有mCAH3的30分钟减少到饱和mCAH3的10秒。最后，使用mGFP标记的mCAH3，我们获得了直接的证据，根据先前的研究和动力学建模，我们得到了CP和mCAH3的复合物对带刺末端有少量但可测量的亲和力的证据。我们得出结论，分离的CARMIL的CAH3结构域，以及可能的完整分子，都具有解帽CP封顶的肌动蛋白细丝的能力。