ISOLATION AND CHARACTERIZATION OF CYTOPLASMIC COFILIN-ACTIN RODS

细胞质肌丝蛋白丝动蛋白-肌动蛋白棒的分离和表征

• 批准号: 8171304
• 负责人: JAMES R BAMBURG
• 金额: $ 0.08万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171304
• 关键词: Actins; Axon; Calcium; Cell Line; Cells; Cognitive deficits; Computer Retrieval of Information on Scientific Projects Database; Cytochalasin D; Cytolysis; Cytoplasm; Dementia; Dendrites; Detergents; Dithiothreitol; Egtazic Acid; F-Actin; Filament; Functional disorder; Funding; Grant; Image; Immunofluorescence Immunologic; In Vitro; Institution; Lead; Length; Measures; Mechanics; Mediating; Needles; Neuroglia; Neurons; Phase; Physiological; Proteins; Proteomics; Research; Research Personnel; Resources; Shapes; Sodium Chloride; Source; Staining method; Stains; Stress; Synapses; Time; United States National Institutes of Health; actin depolymerizing factor; cofilin; jasplakinolide; response; retinal rods; sedimentation equilibrium; tomography; Actins; Axon; Calcium; Cell Line; Cells; Cognitive deficits; Computer Retrieval of Information on Scientific Projects Database; Cytochalasin D; Cytolysis; Cytoplasm; Dementia; Dendrites; Detergents; Dithiothreitol; Egtazic Acid; F-Actin; Filament; Functional disorder; Funding; Grant; Image; Immunofluorescence Immunologic; In Vitro; Institution; Lead; Length; Measures; Mechanics; Mediating; Needles; Neuroglia; Neurons; Phase; Physiological; Proteins; Proteomics; Research; Research Personnel; Resources; Shapes; Sodium Chloride; Source; Staining method; Stains; Stress; Synapses; Time; United States National Institutes of Health; actin depolymerizing factor; cofilin; jasplakinolide; response; retinal rods; sedimentation equilibrium; tomography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca2+, and ATP. Cofilin-GFP-containing rods are stable in 500 mm NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 在应激神经元的轴突和树突形成的Cofilin-actin束（杆）会导致突触功能障碍，并可能介导痴呆症的认知缺陷。杆在非神经元细胞的细胞质中大量形成，响应许多诱导神经元杆的治疗方法。细胞裂解物中的棒在洗涤剂中不稳定，也不添加钙。首先是由ATP止动物诱导并通过机械裂解从细胞中释放的棒，首先是从表达嵌合肌动蛋白 - 脱蛋白因子（ADF）/Cofilin荧光蛋白上通过偏差和平衡沉积物的两个细胞系中分离出来的，然后是神经元和非表达蛋白质蛋白的差异和平衡蛋白。杆含有ADF/Cofilin和肌动蛋白的1：1比。分离的杆在二硫代醇，EGTA，Ca2+和ATP中是稳定的。含Cofilin-GFP的棒在500 mM NaCl中是稳定的，而由内源性蛋白形成的棒在高盐中的稳定性明显较小。由内源性蛋白形成的棒的蛋白质组学分析鉴定出通过细胞的免疫荧光染色检查棒中存在的其他潜在成分。在其形成的所有阶段，只有肌动蛋白和ADF/cofilin在杆上。此外，从这些纯化的蛋白质中，在生理浓度下从体外快速组装了杆，这表明它们是杆形成所需的唯一蛋白质。细胞质杆的形成受到细胞切拉蛋白D和jasplakinolide的抑制。杆形成的时衰变成像显示了随着时间的推移聚集的丰富的小针形杆。通过超微结构断层扫描测量的杆丝长度范围为22至1480 nm。这些结果表明，通过组装Cofilin-Actin亚基形成了棒，然后是ADF/Cofilin饱和F-肌动蛋白的自我关联。