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

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.

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