ISOLATION AND CHARACTERIZATION OF CYTOPLASMIC COFILIN-ACTIN RODS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在应激神经元的轴突和树突中形成的Cofilin-actin束（杆）导致突触功能障碍，并可能介导痴呆症中的认知缺陷。响应于在神经元中诱导杆状物的许多处理，杆状物在非神经元细胞的细胞质中大量形成。细胞裂解物中的棒在洗涤剂中或添加钙的情况下不稳定。首先从表达嵌合肌动蛋白解聚因子（ADF）/cofilin荧光蛋白的两个细胞系中通过OptiPrep梯度上的差异和平衡沉降分离ATP耗竭诱导并通过机械裂解从细胞中释放的杆，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。棒含有1：1比例的ADF/cofilin和肌动蛋白。分离的视杆在二硫苏糖醇、EGTA、Ca 2+和ATP中是稳定的。含有Cofilin-GFP的杆在500 mM NaCl中稳定，而由内源性蛋白形成的杆在高盐中稳定性显著降低。由内源性蛋白质形成的视杆的蛋白质组学分析鉴定了其他潜在组分，其在视杆中的存在通过细胞的免疫荧光染色来检查。只有肌动蛋白和ADF/cofilin是在杆在其形成的所有阶段，此外，在体外从这些纯化的蛋白质在生理浓度的杆的快速组装表明，它们是唯一的蛋白质杆形成所必需的。细胞松弛素D和jasplakin抑制细胞质杆的形成。时间推移成像棒形成显示大量的小针状棒，随着时间的推移合并。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，杆的形成由cofilin-actin亚基的组装，然后由ADF/cofilin饱和的F-肌动蛋白的自缔合。

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.