Triton-soluble phosphovariants of the high molecular weight neurofilament subunit from NB2a/dl cells are assembly-competent: Implications for normal and abnormal neurofilament assembly

Abstract

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 from 35S-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.