Triton-soluble phosphovariants of the high molecular weight neurofilament subunit from NB2a/d1 cells are assembly-competent. Implications for normal and abnormal neurofilament assembly.
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.
Shea
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Laboratories for Molecular Neuroscience, Mailman Research Center, McLean Hospital, Belmont, MA 02178.
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D051379:Mice; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D012995:Solubility
D016900:Neurofilament Proteins; D010750:Phosphoproteins; D011092:Polyethylene Glycols