Triton-soluble phosphovariants of the high molecular weight neurofilament subunit from NB2a/d1 cells are assembly-competent. Implications for normal and abnormal neurofilament assembly.
Triton-soluble phosphovariants of the high molecular weight neurofilament subunit from NB2a/d1 cells are assembly-competent. Implications for normal and abnormal neurofilament assembly.
复制标题
来自 NB2a/d1 细胞的高分子量神经丝亚基的 Triton 可溶性磷酸变体具有组装能力。
DOI：
10.1016/0014-5793(94)80304-8
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发表时间：
1994
期刊：
影响因子：
3.5
通讯作者：
Shea,TB
中科院分区：
文献类型：
--
作者：
Shea,TB
NB2a/dl细胞将含有广泛磷酸化的高（NF - H）分子量亚基的神经丝（NFs）整合到轴突神经突的三不溶性细胞骨架中，这是在二丁基cAMP分化过程中形成的。然而，免疫细胞化学和生化分析表明，一个相当大的（200 kDa） NF - H库的组成性表达和广泛的磷酸化。我们通过无细胞分析来检验这个Triton可溶性NF - H池在无细胞分析中是否具有组装能力。从35s放射性标记的NB2a/dl细胞中提取的Triton可溶性组分与解离的小鼠中枢神经系统（CNS） Triton不溶性细胞骨架（经6m尿素处理解离）一起孵育。经过隔夜透析去除尿素后，低速离心沉淀Triton -不溶性细胞骨架，导致放射性标记的NF - H共同沉淀，表明Triton -可溶性NF - H能够与Triton -不溶性结构相关联。来自NB2a/dl细胞的Triton可溶性、广泛磷酸化的NF - H也能够与纯化的NF - L共组装。高速离心（100,000 × g离心1小时）沉淀任何低聚物，将NB2a/dl细胞中的Triton可溶部分与用6m尿素溶解的纯化NF - L混合。经过隔夜透析去除尿素后，高速离心沉淀NB2a/dl细胞中的NF - L和Triton可溶性NF - H，证明Triton可溶性NF - H变体具有组装能力。这些数据表明NF - H变异代表NF组装的前体，并表明它们在NB2a/dl细胞内的组装必须受到时间和空间的调节。
NB2a/dl cells incorporate neurofilaments (NFs) containing extensively phosphorylated high (NF‐H) molecular weight subunits into the Tritoninsoluble cytoskeleton of axonal neurites elaborated during differentiation with dibutyryl cAMP. However, immunocytochemical and biochemical analyses demonstrate the constitutive expression and extensive phosphorylation of a sizeable pool of (200 kDa) NF‐H. We examined by cell‐free analyses whether or not this Triton‐soluble NF‐H pool was assembly‐competent in cell‐free analyses. Triton‐soluble fractions from35S‐radiolabeled NB2a/dl cells were incubated with dissociated mouse CNS Triton‐insoluble cytoskeletons that had been dissociated by treatment with 6 M urea. Following overnight dialysis to remove urea, low‐speed centrifugation to sediment Triton‐insoluble cytoskeletons resulted in the co‐sedimentation of radiolabeled NF‐H, indicating that Triton‐soluble NF‐H was capable of association with Triton‐insoluble structures. Triton‐soluble, extensively phosphorylated NF‐H from NB2a/dl cells was also capable of co‐assembling with purified NF‐L. Following high‐speed centrifugation (100,000 × g for 1 h) to sediment any oligomeric assemblies, the Triton‐soluble fraction from NB2a/dl cells was mixed with purified NF‐L that had been solubilized by 6 M urea. Following overnight dialysis to remove urea, high‐speed centrifugation sedimented both NF‐L and Triton‐soluble NF‐H from NB2a/dl cells, demonstrating that Triton‐soluble NF‐H variants are assembly‐competent. These data suggest that NF‐H variants represent precursors for NF assembly, and indicate that their assembly within NB2a/dl cells must be under temporal and spatial regulation.