ISOLATION AND CHARACTERIZATION OF CYTOPLASMIC COFILIN-ACTIN RODS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在应激神经元的轴突和树突中形成的粘连蛋白-肌动蛋白束(ROD)可导致突触功能障碍，并可能介导痴呆的认知障碍。在非神经元细胞的细胞质中形成大量的杆状结构，以响应许多诱导神经元内杆状结构的处理。细胞裂解物中的杆状物质在洗涤剂或添加钙的情况下不稳定。首先通过OptiPrep梯度分离和平衡沉淀法从两个表达嵌合肌动蛋白解聚因子(ADF)/cofilin荧光蛋白的细胞系中分离到由ATP耗竭诱导的、通过机械裂解从细胞中释放出来的杆状蛋白，然后从仅表达内源性蛋白的神经元和非神经元细胞中分离出杆状蛋白。视杆含有ADF/Cofilin和肌动蛋白，比例为1：1。分离的杆子在二硫苏糖醇、EGTA、钙离子和三磷酸腺苷中是稳定的。含有cofilin-GFP的杆状物在500 mM的氯化钠中是稳定的，而由内源蛋白形成的杆状物在高盐中明显不稳定。对由内源蛋白形成的杆状细胞进行的蛋白质组学分析确定了其他潜在的成分，这些成分的存在通过细胞的免疫荧光染色进行了检测。在杆形成的所有阶段，只有肌动蛋白和ADF/cofilin存在于杆中；此外，这些纯化蛋白在生理浓度下在体外快速组装杆表明，它们是杆形成所必需的唯一蛋白质。细胞松弛素D和茉莉酸激内酯抑制细胞质杆状的形成。棒状物形成的时间推移成像显示，随着时间的推移，大量细小的针状棒状物结合在一起。超微结构断层扫描测量的杆状细丝长度为22~1480 nm。这些结果表明，杆状细胞是由粘连蛋白-肌动蛋白亚单位组装而成，然后是ADF/粘连蛋白饱和的F-肌动蛋白的自结合。

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.