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  • Title: Triton-soluble phosphovariants of the high molecular weight neurofilament subunit from NB2a/d1 cells are assembly-competent. Implications for normal and abnormal neurofilament assembly.
    Author: Shea TB.
    Journal: FEBS Lett; 1994 Apr 25; 343(2):131-6. PubMed ID: 8168617.
    Abstract:
    NB2a/d1 cells incorporate neurofilaments (NFs) containing extensively phosphorylated high (NF-H) molecular weight subunits into the Triton-insoluble 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 from 35S-radiolabeled NB2a/d1 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/d1 cells was also capable of co-assembling with purified NF-L. Following high-speed centrifugation (100,000 x g for 1 h) to sediment any oligomeric assemblies, the Triton-soluble fraction from NB2a/d1 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/d1 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/d1 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/d1 cells must be under temporal and spatial regulation.
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